Integrated semiconductor bioarray

ABSTRACT

A biosensor array, system and method for affinity based assays that are able to simultaneously obtain high quality measurements of the binding characteristics of multiple analytes, and that are able to determine the amounts of those analytes in solution. The invention also provides a fully integrated bioarray for detecting real-time characteristics of affinity based assays.

CROSS-REFERENCE

This application claims priority to U.S. Provisional Application60/840,060, filed Aug. 24, 2006, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Biosensor detection systems take advantage of the selective interactionand binding (affinity) of certain biological molecules to identifymolecular structures and furthermore measure levels of differentanalytes such as toxins, polymers, hormones, DNA strands, proteins, andbacteria. Affinity-based biosensors exploit selective binding andinteraction of certain bio-molecules (recognition probes) to detectspecific target analytes in biological samples. The performance ofbiosensors in terms of signal to noise ratio and dynamic range isgenerally constrained by the characteristics of the molecularrecognition layer which captures the target analytes, and not by thetransducer and read-circuitry. An advantage of biosensors is theircapability to be implemented in parallel and in an array format.Biosensors in parallel may compensate for limited detection performance.Presently, densely packed biosensor arrays which detect thousands ofdifferent analytes simultaneously (microarrays) are popular in Genomics,Proteomics, molecular diagnostics, and systems biology.

The essential role of the biosensor platforms and the parallel andminiaturized versions of them as microarrays are to exploit specificbindings of the probe-target complexes to produce detectable signals,which correlate with the presence of the targets and conceivably theirabundance.

Many of the microarrays currently used in biological and medicalresearch are DNA microarrays, in which the probe that is spotted orsynthesized onto a solid surface is DNA. However, in addition to nucleicacids, microarray technologies are applicable to other types ofbiochemical compounds and analytes that can be immobilized on solidsurfaces, such as proteins, carbohydrates, and lipids. Microarrays canbe used to study the interactions between compounds of a same ordifferent type (for example, protein-protein interactions, orprotein-carbohydrate interactions).

Beside all the uncertainties within the measurement results, there isalso a question in microarrays and most affinity-based biosensorsystems, and that is of the necessary incubation time (hybridizationtime for DNA microarrays). Since the incubation kinetics in themicroarrays experiments is a function of analyte diffusion, reactionchamber size, temperature and binding kinetics of every analyte species,as well as the unknown analyte concentrations, the settling time of thesystem is quite complex and unpredictable. Although all these questionscan, to some extent, be empirically addressed, they are still majorimpediments in microarray technology and platform-to-platforminconsistencies can be caused by them.

In conventional fluorescent-based microarrays and other extrinsicreporter-based (label-based) biosensors assays, the detection ofcaptured analytes is usually carried out after the incubation step. Insome cases, proper fluorescent and reporter intensity measurements arecompromised in the presence of a large concentration of floating(unbound) labeled species in the incubation solution, whose signal canoverwhelm the target-specific signal from the captured targets. When theincubation is ceased and the solution is removed from the surface of thearray, the washing artifacts often occur that make the analysis of thedata even more challenging. Thus there exists a need for affinity basedsensors that are able to simultaneously obtain high quality measurementsof the binding characteristics of multiple analytes, and that are ableto determine the amounts of those analytes in solution.

The emerging high-through screening and point-of-care (PoC) diagnosticsapplications demand the integration of the biochemical part (assays) ofthe detection platform with the transducer and the detection circuitry.A microarray is desired in the art that offers compact andcost-efficient solutions with a high production yield and robustfunctionality.

SUMMARY OF THE INVENTION

In an aspect of the invention, a biosensor array comprises, in order, amolecular recognition layer, an optical layer and a sensor layerintegrated in a sandwich configuration. The molecular recognition layercan comprise an open surface and a plurality of different probesattached at different independently addressable locations to the opensurface. The molecular recognition layer can transmit light to theoptical layer. The optical layer comprises an optical filter layer,wherein the optical layer transmits light from the molecular recognitionlayer to the sensor layer. The sensor layer comprises an array ofoptical sensors that detect the filtered light transmitted through theoptical layer.

In the biosensor array, the sensor layer can comprise embedded detectioncircuitry connected to the array of optical sensors. In anotherembodiment, the sensor layer comprises embedded detection and signalprocessing circuitry connected to the optical sensors.

The sensor layer can comprise a photodiode array.

In an embodiment, the sensor layer is a semiconductor device. Thesemiconductor device can be a silicon semiconductor device. Examples ofa semiconductor device used as the sensor layer of an embodiment of theinvention include, but are not limited to, a charge coupled device(CCD), a complementary metal oxide semiconductor (CMOS), and a digitalsignal processor.

In an embodiment, the biosensor array comprises an integrated in-pixelphotocurrent detector. The detector can be capacitive transimpedanceamplifier (CTIA).

In another embodiment, the biosensor array has an in-pixel analog todigital converter.

In an embodiment, the optical layer of the biosensor array furthercomprises an optical coupling layer between the optical filter layer andthe molecular recognition layer. The optical coupling layer can comprisea plurality of optical waveguides. The optical coupling layer can alsocomprise a fiber-optic faceplate.

In an embodiment, the optical layer is 2 μm to 20 cm thick. In a furtherembodiment, the optical layer is 5 μm to 1 cm thick.

In another embodiment, the optical layer can provide thermal insulationbetween the sensor layer and the molecular recognition layer.

The optical filter layer of the optical layer can comprise a multilayerdielectric. In an embodiment, the optical filter has a passband or astopband with a bandwidth of about 10 nm to 20 nm. In anotherembodiment, the passband or stopband is in the range of 400 nm to 800nm.

In another embodiment, the optical filter attenuates fluorescentexcitation light by 10² to 10⁷. In a further embodiment, the opticalfilter attenuates fluorescent excitation light by 10³ to 10⁵.

The biosensor array can comprise at least one optical sensorcorresponding to one independently addressable location comprising aprobe. In another embodiment, more than one optical sensor correspondsto one independently addressable location. In yet another embodiment,about 10 to about 1000 optical sensors correspond to one independentlyaddressable location. Different optical sensors corresponding to oneindependently addressable location can measure different wavelengths oflight.

In an embodiment, the molecular recognition layer comprises 2 to1,000,000 probes. The independently addressable regions of the molecularrecognition layer can comprise probes comprising fluorescent moieties.In an embodiment, the fluorescent moieties are capable of being quenchedupon binding of an analyte comprising a quencher. In another embodiment,the fluorescent moieties are bound to the probes. In yet anotherembodiment, the fluorescent moieties are bound to the surface of thearray, but are not covalently bound to the probes.

In an embodiment, the probes comprise nucleic acids. In anotherembodiment, the probes comprise proteins.

In another aspect of the invention, a biosensor system comprises a fullyintegrated biosensor array comprising (a) a solid substrate comprisingan array of semiconductor-based optical sensors, (b) an optical filterlayer in contact with the solid substrate, (c) an optical coupling layerin contact with the optical filter layer, and (d) a molecularrecognition layer in contact with the optical coupling layer andcomprising a plurality of different probes, with different probesimmobilized to the surface at a different addressable locations. Thesystem further comprises a light source that directs light to thebiosensor array, a fluidic chamber for holding fluid in contact with thebiosensor array, a temperature controller for controlling thetemperature of the fluid and/or the molecular recognition layer, and aninterface that connects to the biosensor array to allow electroniccommunication to and from the array.

In an embodiment, the biosensor system further comprises a computer withsoftware for process control and data collection and analysis. Thesystem can comprise a plurality of biosensor arrays that use the samecomputer.

In an embodiment, the system comprises from 2 to 12 biosensor arrays.

In an embodiment, the different addressable locations of the fullyintegrated biosensor array comprise quenchable tags.

A system of the invention can comprise control circuitry that enablesusers to activate readout circuits and sensors on the array. In anembodiment, the system comprises control circuitry to specifytemperature, mixing, fluorescent wavelength, fluorescent excitationpower, pressure, and/or humidity. In another embodiment, at least partof the control circuitry is in the biosensor array.

In an embodiment, the system comprises decoder circuitry to select whichof a plurality of sensors to be used. In a further embodiment, at leastpart of the decoder circuitry is in the biosensor array.

In an embodiment, the light source of the system is fixed, andilluminates all of the addressable locations.

In an aspect of the invention, a method comprises contacting a fluidcontaining a target analyte with a fully integrated biosensor array ofthe invention, illuminating the biosensor, detecting fluorescence on thebiosensor, and correlating the detected fluorescence with binding of thetarget analyte.

In another aspect, a method comprises (a) contacting a fluid volumecomprising a plurality of different analytes with a solid substratecomprising a plurality of different probes at independently addressablelocations, wherein the probes are capable of specifically binding to theanalytes, and (b) measuring signals at multiple time points while thefluid volume is in contact with the substrate, wherein the signalsmeasured at multiple time points can be correlated with the amount ofbinding of the analytes with the probes. The solid substrate comprisesan array of optical transducers, wherein at least one optical transducercorrelates to one independently addressable location. In an embodiment,the method further comprises (c) using the signals measured at multipletime points to determine the concentration of an analyte in the fluidvolume.

In an embodiment, a change in the signals with time correlates with theamount of the analytes bound to the probes.

In yet another aspect of the invention, a method comprises (a)performing a nucleic acid amplification on two or more nucleotidesequences to produce two or more amplicons in a fluid wherein the arraycomprises a solid surface with a plurality of nucleic acid probes atindependently addressable locations. The solid substrate comprises anarray of optical transducers, wherein at least one optical transducercorrelates to one independently addressable location. The method alsocomprises (b) measuring the hybridization of the amplicons to the two ormore nucleic acid probes while the fluid is in contact with the array toobtain an amplicon hybridization measurement. In an embodiment of themethod of the invention, the method further comprises using the ampliconhybridization measurement to determine the concentration of theamplicons in the fluid. In another embodiment, the method furthercomprises using the amplicon hybridization measurement to determine theoriginal amount of nucleotide sequences.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates an example of a conventional microarray of the priorart.

FIG. 2 displays an example of prior art microarray technology based uponfluorescent detection.

FIG. 3 illustrates an integrated biosensor array of the invention.

FIG. 4 demonstrates a passive pixel sensor (PPS), an active pixel sensor(APS), and a digital pixel sensor (DPS) for use as the pixelarchitecture of a CMOS image sensor array.

FIG. 5 shows an example of a CMOS embodiment of the present invention.

FIG. 6 illustrates an embodiment of the invention comprising a fiberoptic faceplate (FOF).

FIG. 7 shows a block diagram of some of the components of systems of thepresent invention.

FIG. 8 illustrates an embodiment of a system comprising an assay kit, adisposable sensor chip, a computer chip reader and software forexecuting a method of the invention.

FIG. 9 illustrates the absorption and emission spectra of Cy3 molecule.

FIG. 10 demonstrates an example design of a system of the inventioncomprising a capacitive transimpedance amplifier (CTIA).

FIG. 11 shows the die photo and the I/O pin allocation of an integrated7 by 8 pixel bio sensor array.

DETAILED DESCRIPTION OF THE INVENTION

The devices, systems, and methods disclosed herein concern integratedbiosensor arrays. One aspect of the invention is a fully integratedarray which comprises a molecular recognition layer, an optical layer,and a sensor layer in a sandwich configuration. The biosensor array iscapable of measuring the presence, amount, and binding characteristicsof multiple analytes in solution by measuring the binding of theanalytes to probes in the molecular recognition layer which is on anopen surface of the biosensor array. The molecular recognition layercomprises a plurality of probes that are located on discrete,independently addressable regions. The optical layer comprises anoptical filter layer such as a multilayer dielectric which selectivelyallows light within a given wavelength range to pass through to thesensor array. The optical layer can also comprise an optical couplinglayer, for example a fiber optic faceplate that guides the light fromthe molecular layer to the optical filter layer and/or the sensor array.The sensor layer comprises an array of sensors, wherein typically thesensors correspond to the independently addressable regions of probes.

The sensor array can be a semiconductor based photodiode array which canbe created in a silicon substrate using, for example, a CMOS process.Where the diode array is made by conventional silicon processing such asCMOS, additional circuitry can be incorporated into the array to enhancethe signal processing capability of the array and to improve dataquality. The additional circuitry can be incorporated into each pixel ofthe array, for example comprising an in-pixel photocurrent detector, orcan be placed on other portions of the silicon chip. In some cases, thesensor array has multiple sensors, for example, 10 to 1000 sensors whichcorrespond to the same independently addressable region of probe. Insome cases, different sensors that correspond to the same addressableregion of probe correspond to different regions of wavelength (differentcolors). The biosensor array could have a few addressable regions, suchas 3 to 20, or could have a large number of addressable regions from1,000 to 100,000 or more.

The biosensor arrays can be incorporated into a system comprising alight source for illuminating the sample, a fluidic chamber for holdingthe fluid in contact with the molecular recognition layer, a temperaturecontroller for controlling hybridization conditions, an interface forallowing electronic communication with the array. The system can in somecases measure multiple biosensor arrays simultaneously.

The biosensor arrays of the present invention can be used for thedetection of the binding of analytes in real-time. By measuringReal-time measurement of the kinetics of multiple binding events allowsfor an accurate and sensitive determination of binding characteristicsor of analyte concentration for multiple species simultaneously. Theabundance of target analytes in a sample can be evaluated by thereal-time detection of target-probe binding events. In some embodiments,real-time microarray (RT-μArray) detection systems measure theconcentration of the target analytes by analyzing the binding ratesand/or the equilibrium concentration of the captured analytes in asingle and/or plurality of spots. The measurement of analyteconcentration during binding can avoid errors introduced in the washingand drying process used in many conventional microarrays. Measuringduring binding also has the advantage of taking less time than waitingfor saturation of binding before measuring analyte binding.

The molecular recognition layer can comprise fluorescent entities thatare attached to the surface that are quenched by analytes comprisingquenchers in solution. This method can be advantageous for measuringbinding in the presence of solution because the analytes comprisingquenchers can be designed so as to contribute minimally to backgroundfluorescence. The fluorescent entities can be bound directly to theprobe or can be bound to the surface in the vicinity of the probe.

The integrated biosensor arrays of the invention can be used to measurethe concentration or binding characteristics of many types ofprobe-analyte binding pairs including proteins and nucleic acids. Themeasurement of nucleic acid hybridization on the biosensor arrays of theinvention can be used to perform genomic and genetic expression analysisaccurately and rapidly on many nucleotide sequences simultaneously. Thebiosensor arrays of the present invention can be used for accuratediagnostic and medical testing.

Biosensor Array

The biosensor array (bioarray) of the present invention is an integratedarray comprising a molecular recognition layer, an optical layer, and asensor layer.

A technique to measure the amount of captured analytes in microarrays(i.e., transduction method) is based on fluorescence spectroscopy.Previous microarray technology and biosensors have often been focused onsystems with electrochemical transducers or partially integratedfluorescent and bioluminescence detectors. An example of a conventionalmicroarray of the prior art is shown in FIG. 1. The system consists ofan affinity-based assay located in contact with an array containingindependently addressable probe locations. After a step of incubation toallow an analytes in the affinity-based assay to bind with anappropriate probe, the binding affinity can be detected by a sensor.Many systems of the prior art are two step systems as illustrated inFIG. 1.

In many embodiments, the devices, systems, and methods of the inventionutilize fluorescent detection. An example of prior art microarraytechnology based upon fluorescent detection is illustrated in FIG. 2. Amicroarray on a glass slide contains independently addressable locationscontaining capturing probes, as illustrated. A fluorescent label isattached to a target analyte. An incubation step is carried out to allowthe target analyte to bind to the capturing probe. After the incubationstep is complete, a fluorescent image can be taken of the microarray andthe different independently addressable locations emit different signalscorresponding to the amount of analyte bound on the location. In thepresent invention, the optical sensors are integrated into the substrateonto which capturing probes are bound.

An aspect of the invention is a fully integrated biosensor arraycomprising, in order, a molecular recognition layer, an optical layerand a sensor layer integrated in a sandwich configuration. The molecularrecognition layer comprises an open surface and a plurality of differentprobes attached at different independently addressable locations to theopen surface. The molecular recognition layer can also transmit light tothe optical layer. The optical layer comprises an optical filter layer,wherein the optical layer transmits light from the molecular recognitionlayer to the sensor layer. The transmittal of light between layers canbe filtered by the optical layer. The sensor layer comprises an array ofoptical sensors that detects the filtered light transmitted through theoptical layer.

An integrated biosensor array of the invention can measure binding ofanalytes in real-time. FIG. 3 illustrates an embodiment of a device ofthe invention. An integrated biosensor microarray that can detectbinding kinetics of an assay is in contact with an affinity-based assay.The biosensor array comprises a molecular recognition layer comprisingbinding probes in optical communication a sensor for detecting bindingto the probes in real-time.

An integrated fluorescent-based microarray system for real-timemeasurement of the binding of analyte to a plurality of probes thatincludes the capturing probe layer, fluorescent emission filter, andimage sensor can be built using a standard complementary metal-oxidesemiconductor (CMOS) process.

The devices of the invention can measure binding of analytes to aplurality of probes on surface in “real-time”. As used herein inreference to monitoring, measurements, or observations of binding ofprobes and analytes of this invention, the term “real-time” refers tomeasuring the status of a binding reaction while that reaction isoccurring, either in the transient phase or in biochemical equilibrium.Real-time measurements are performed contemporaneously with themonitored, measured, or observed binding events, as opposed tomeasurements taken after a reaction. Thus, a “real-time” assay ormeasurement contains not only the measured and quantities result, suchas fluorescence, but expresses this at various time points, that is, inhours, minutes, seconds, milliseconds, nanoseconds, etc. “Real-time”includes detection of the kinetic production of signal, comprisingtaking a plurality of readings in order to characterize the signal overa period of time. For example, a real-time measurement can comprise thedetermination of the rate of increase or decrease in the amount ofanalyte bound to probe.

Real-time measurement can include the measurement of the bindingkinetics to characterize binding of multiple probes and analytes insolution. In some cases, binding reaction refers to the concurrentbinding reactions of multiple analytes and probes, and in other cases,the term binding reaction refers to the reaction between a single probewith a single analyte. The meaning will be clear from the context ofuse. The kinetic measurements can be expressed as the amount of analytebound to the probe as a function of time. The binding kinetics canprovide information about the characteristics of the probe-analytebinding such as the strength of binding, the concentration of analyte,the competitive binding of an analyte, the density of the probes, or theexistence and amount of cross-hybridization.

In order to determine binding kinetics, the signal at multiple timepoints must be determined. The signal at least two time points isrequired. In most cases, more than two time points will be desired inorder to improve the quality of the kinetic information. In someembodiments the signal at, 2-10, 10-50, 50-100, 100-200, 200-400,400-800, 800-1600, 1600-3200, 3200-6400, 6400-13000, or higher than13,000 time points will be measured. One of ordinary skill in the artcan determine the effective number of points for a given embodiment.

The frequency at which the signal is measured will depend on thekinetics of the binding reaction or reactions that are being monitored.As the frequency of measurements gets lower, the time betweenmeasurements gets longer. One way to characterize a binding reaction isto refer to the time at which half of the analyte will be bound(t_(1/2)). The binding reactions of the invention can have a (t_(1/2))from on the order of milliseconds to on the order of hours, thus thefrequency of measurements can vary by a wide range. The time betweenmeasurements will generally not be even over the time of the bindingreaction. In some embodiments, a short time between of measurements willbe made at the onset of the reaction, and the time between measurementswill be longer toward the end of the reaction. One advantage of thepresent invention is the ability to measure a wide range of bindingrates. A high initial frequency of measurements allows thecharacterization of fast binding reactions which may have higherbinding, and lower frequency of measurements allows the characterizationof slower binding reactions. For example, points can initially bemeasured at a time between points on the order of a microsecond, thenafter about a millisecond, points can be measured at a time betweenpoints on the order of a millisecond, then after about a second, timepoints can be measured at a time between points on the order of asecond. Any function can be used to ramp the change in measurementfrequency with time. In some cases, changes in the reaction conditions,such as stringency or temperature changes will be made during areaction, after which it may be desirable to change the frequency ofmeasurements to measure the rates of reaction which will be changed bythe change in reaction condition.

In some embodiments, a probe will have substantially no analyte bound toit at the beginning of the binding reaction, then the probe will beexposed to a solution containing the analyte, and the analyte will beginto bind, with more analyte bound to the probe with time. In some cases,the reaction will reach saturation, the point at which all of theanalyte that is going to bind has bound. Generally, saturation willoccur when a reaction has reached steady state. At steady state, in agiven time period, just as many analytes are released as new analytesare bound (the on rate and off rate are equal). In some cases, with verystrong binding, where the off-rate for the analyte is essentially zero,saturation will occur when substantially all of the analyte that canbind to the probe will have bound, has bound. Thus, while it isadvantageous to measure a change in signal with time that can becorrelated with binding kinetics, the measurement of a signal that doesnot change with time also provides information in the context of areal-time experiment, and can also be useful in the present invention.For example, in some cases the absence of a change in the signal willindicate the level of saturation. In other cases the absence of a changein signal can indicate that the rate of the reaction is very slow withrespect to the change in time measured. It is thus a beneficial aspectof this invention to measure binding event in real time both wheresignals change with time and where the signals do not change with time.

One aspect of the methods of the present invention is the measurement ofconcentration of an analyte from the measurement of binding kinetics.Since analyte binding rate can be concentration-dependant, we canestimate the analyte abundance in the sample solution using bindingrates.

One aspect of the present invention is the determination of the bindingof analyte to probe by measuring the rate near the beginning of thereaction. In addition to providing a more reliable estimate of C,measurement near the beginning of the reaction can shorten the time thatis required to measure analyte binding over the time required formeasuring binding from saturation. In some embodiments of the invention,the binding is measured during the time for less than about the first0.1, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 18, 20, 25, 30, 40, 50, 60,70, 80, or 90 percent of the analyte to bind as compared to the amountof analyte bound at saturation. In some embodiments, the bindingkinetics are determined in a time for less than about the first 20% ofthe analyte to bind. In some embodiments, the binding kinetics aredetermined in a time for less than about the first 1-2% of the analyteto bind.

Molecular Recognition Layer

A molecular recognition layer of the present invention comprises an opensurface of the biosensor array.

A molecular recognition layer of the invention comprises a plurality ofprobes located at independently addressable locations for detectingdifferent analytes. The probes can specifically bind with analytes insolution in order to determine the concentration and bindingcharacteristics of the analytes. The probes can be used to detectanalytes including, but not limited to, polymers, hormones, DNA strands,proteins, and bacteria. In some embodiments, the molecular recognitionlayer comprises 2 to 1,000,000 probes.

In another embodiment, the independently addressable regions compriseprobes that are fluorescent moieties. The fluorescent moieties can alsobe bound to the probes. The probe-bound fluorescent moieties can bequenched by analytes comprising quenchers, such that the quenching offluorescence can be used to determine the concentration of analyte insolution. The molecular recognition layer can transmit light through anoptical layer to a sensor layer in a biosensor array of the invention.Often, the light is transmitted by fluorescence from the interaction andbinding of an analyte to a probe through the optical layer to the sensorlayer.

In some embodiments, the fluorescent moieties are bound to the surfaceof the array, and are not covalently bound to the probes. Surface boundfluorescent moieties can be quenched by analytes in solution comprisingquenchers and thus used to determine the concentration of analyte insolution even where the fluorescent moiety is not bound to the probeitself.

In an embodiment, the probes of the molecular recognition layer arenucleic acids. The probes can also be proteins.

In other embodiments, the RT-μArray is placed onto a transducer array.The distance of the probes to the transducer array in such a setup canbe from 1 mm to 0.1 μm. In some embodiments, the distance is about 20 μmto 2 μm.

The arrays of the present invention comprise probes which comprise amolecular recognition layer. The layer may be biological, nonbiological,organic, inorganic, or a combination of any of these. The surface onwhich the probes reside can exist as particles, strands, precipitates,gels, sheets, tubing, spheres, containers, capillaries, pads, slices,films, plates, slides, semiconductor integrated chips, etc. The surfaceis preferably flat but may take on alternative surface configurations.For example, the molecular recognition layer may occur on raised ordepressed regions on which synthesis or deposition takes place. In someembodiments, the molecular recognition layer will be chosen to provideappropriate light-absorbing characteristics. For example, the layer maybe a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge,GaAs, GaP, SiO₂, SiN₄, modified silicon, or any one of a variety of gelsor polymers such as (poly)tetrafluoroethylene,(poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinationsthereof.

The surface can be a homogeneous solid and/or unmoving mass much largerthan the capturing probe where the capturing probes are confined and/orimmobilized within a certain distance of it. In certain embodiments, thesurface is planar with roughness of 0.1 nm to 100 nm, but typicallybetween 1 nm to 10 nm. In other embodiments the surface can be a poroussurface with roughness of larger than 100 nm. In other embodiments, thesurface can be non-planar. Examples of non-planar surfaces are sphericalmagnetic beads, spherical glass beads, and solid metal and/orsemiconductor and/or dielectric particles.

The molecular recognition layer is typically in contact with fluid thatis optically transparent to an excitation source light. After excitationof the molecular recognition layer, the layer can emit an opticalwavelength that can be detected.

In some embodiments, the molecular recognition layer may be bound to aglass surface when the optical layer comprises glass. The molecularrecognition layer can also be bound to silica, silicon, plastic, metal,metal-alloy, anopore, polymeric, and nylon. The surfaces to which themolecular recognition layer is bound can be treated with a layer ofchemicals prior to attaching probes to enhance the binding or to inhibitnon-specific binding during use. For example, glass surfaces can becoated with self-assembled monolayer (SAM) coatings, such as coatings ofas aminoalkyl silanes, or of polymeric materials, such as acrylamide andproteins.

Probes can be attached covalently (but non-covalent attachment methodscan also be used). A number of different chemical surface modifiers canbe added to the surface to attach the probes. Examples of chemicalsurface modifiers include N-hydroxy succinimide (NHS) groups, amines,aldehydes, epoxides, carboxyl groups, hydroxyl groups, hydrazides,hydrophobic groups, membranes, maleimides, biotin, streptavidin, thiolgroups, nickel chelates, photoreactive groups, boron groups, thioesters,cysteines, disulfide groups, alkyl and acyl halide groups, glutathiones,maltoses, azides, phosphates, and phosphines. Glass slides with suchchemically modified surfaces are commercially available for a number ofmodifications. These can easily be prepared for the rest, using standardmethods (Microarray Biochip Technologies, Mark Schena, Editor, March2000, Biotechniques Books).

In some embodiments, surfaces that are reactive to probes comprisingamines are used. An advantage of this reaction is that it is fast, withno toxic by-products. Examples of such surfaces include NHS-esters,aldehyde, epoxide, acyl halide, and thio-ester. Most proteins, peptides,glycopeptides, etc. have free amine groups, which will react with suchsurfaces to link them covalently to these surfaces. Nucleic acid probeswith internal or terminal amine groups can also be synthesized, and arecommercially available (e.g., from IDT or Operon). Thus, nucleic acidscan be bound (e.g., covalently or non-covalently) to surfaces usingsimilar chemistries.

The surfaces to which the probes are bound need not be reactive towardsamines, but can be easily converted into amine-reactive surfaces withcoatings. Examples of coatings include amine coatings (which can bereacted with bis-NHS cross-linkers and other reagents), thiol coatings(which can be reacted with maleimide-NHS cross-linkers, etc.), goldcoatings (which can be reacted with NHS-thiol cross linkers, etc.),streptavidin coatings (which can be reacted with bis-NHS cross-linkers,maleimide-NHS cross-linkers, biotin-NHS cross-linkers, etc.), and BSAcoatings (which can be reacted with bis-NHS cross-linkers, maleimide-NHScross-linkers, etc.). Alternatively, the probes, rather than the opensurface, can be reacted with specific chemical modifiers to make themreactive to the respective surfaces.

A number of other multi-functional cross-linking agents can be used toconvert the chemical reactivity of one kind of surface to another. Thesegroups can be bifunctional, tri-functional, tetra-functional, and so on.They can also be homo-functional or hetero-functional. An example of abi-functional cross-linker is X—Y—Z, where X and Z are two reactivegroups, and Y is a connecting linker. Further, if X and Z are the samegroup, such as NHS-esters, the resulting cross-linker, NHS—Y—NHS, is ahomo-bi-functional cross-linker and would connect an amine surface withan amine-group containing molecule. If X is NHS-ester and Z is amaleimide group, the resulting cross-linker, NHS—Y-maleimide, is ahetero-bi-functional cross-linker and would link an amine surface (or athiol surface) with a thio-group (or amino-group) containing probe.Cross-linkers with a number of different functional groups are widelyavailable. Examples of such functional groups include NHS-esters,thio-esters, alkyl halides, acyl halides (e.g., iodoacetamide), thiols,amines, cysteines, histidines, di-sulfides, maleimide, cis-diols,boronic acid, hydroxamic acid, azides, hydrazines, phosphines,photoreactive groups (e.g., anthraquinone, benzophenone), acrylamide(e.g., acrydite), affinity groups (e.g., biotin, streptavidin, maltose,maltose binding protein, glutathione, glutathione-S-transferase),aldehydes, ketones, carboxylic acids, phosphates, hydrophobic groups(e.g., phenyl, cholesterol), etc. Such cross-linkers can be reacted withthe surface or with the probes or with both, in order to conjugate aprobe to a surface.

Other alternatives include thiol reactive surfaces such as acrydite,maleimide, acyl halide and thio-ester surfaces. Such surfaces cancovalently link proteins, peptides, glycopeptides, etc., via a (usuallypresent) thiol group. Nucleic acid probes containing pendantthiol-groups can also be easily synthesized.

Alternatively, one can modify glass surfaces with molecules such aspolyethylene glycol (PEG), e.g. PEGs of mixed lengths.

Other surface modification alternatives (such as photo-crosslinkablesurfaces and thermally cross-linkable surfaces) are known to thoseskilled in the art. Some technologies are commercially available, suchas those from Mosiac Technologies (Waltham, Mass.), Exiqon™ (Vedbaek,Denmark), Schleicher and Schuell (Keene, N. H.), Surmodics™ (St. Paul,Minn.), Xenopore™ (Hawthorne, N. J.), Pamgene (Netherlands), Eppendorf(Germany), Prolinx (Bothell, Wash.), Spectral Genomics (Houston, Tex.),and Combimatrix™ (Bothell, Wash.).

Surfaces other than glass are also suitable binding the probes of themolecular recognition layer. For example, metallic surfaces, such asgold, silicon, copper, titanium, and aluminum, metal oxides, such assilicon oxide, titanium oxide, and iron oxide, and plastics, such aspolystyrene, and polyethylene, zeolites, and other materials can also beused. In some embodiments, the layers of these materials will have to bemade thin, e.g. less than about 100 nm in order to allow thetransmission of light. The devices can also be prepared on LED (LightEmitting Diode) and OLED (Organic Light Emitting Diode) surfaces. Anarray of LEDs or OLEDs can be used at the base of a probe array. Anadvantage of such systems is that they provide easy optoelectronic meansof result readout. In some cases, the results can be read-out using anaked eye.

Probes can be deposited onto the substrates, e.g., onto a modifiedsurface, using either contact-mode printing methods using solid pins,quill-pins, ink-jet systems, ring-and-pin systems, etc. (see, e.g., U.S.Pat. Nos. 6,083,763 and 6,110,426) or non-contact printing methods(using piezoelectric, bubble-jet, syringe, electro-kinetic, mechanical,or acoustic methods. Devices to deposit and distribute probes ontosubstrate surfaces are produced by, e.g., Packard Instruments. There aremany other methods known in the art. Preferred devices for depositing,e.g., spotting, probes onto substrates include solid pins or quill pins(Telechem/Biorobotics).

In other embodiments, the molecular recognition layer is manufacturedthrough the in-situ synthesis of the probes. This in-situ synthesis canbe achieved using phosphoramidite chemistry and/or combinatorialchemistry. In some cases, the deprotection steps are performed byphotodeprotection (such as the Maskless Array Synthesizer (MAS)technology, (NimbleGen, or the photolithographic process, byAffymetrix). In other cases, deprotection can be achievedelectrochemically (such as in the Combimatrix procedure). Microarraysfor the present invention can also be manufactured by using the inkjettechnology (Agilent).

For the arrays of the present invention, the plurality of probes may belocated in one addressable region and/or in multiple addressable regionson the open surface of the molecular recognition layer. In someembodiments the molecular recognition layer has about 2, 3, 4, 5, 6, or7-10, 10-50, 50-100, 100-500, 500-1,000, 1,000-5,000, 5,000-10,000,10,000-50,000, 50,000-100,000, 100,000-500,000, 500,000-1,000,000 orover 1,000,000 addressable regions with probes.

The spots may range in size from about 1 nm to about 10 mm, in someembodiments from about 1 to about 1000 micron and more in someembodiments from about 5 to about 100 micron. The density of the spotsmay also vary, where the density is generally at in some embodimentsabout 1 spot/cm², in some embodiments at least about 100 spots/cm² andin other embodiments at least about 400 spots/cm², where the density maybe as high as 10⁶ spots/cm² or higher.

The shape of the spots can be square, round, oval or any other arbitraryshape.

In some embodiments it is also useful to have addressable regions whichdo not contain probe, for example, to act as control spots in order toincrease the quality of the measurement, for example, by using bindingto the spot to estimate and correct for non-specific binding.

Sensor Layer

The sensor layer comprises an array of optical sensors. The sensor arraydetects the light that is transmitted at the molecular recognitionlayer. The light that reaches the sensor array will have passed throughthe optical layer which comprises an optical filter layer that onlyallows a portion of the spectrum of light and/or certain wavelengths toreach the sensors.

The array of optical sensors can be utilized to detect analyteinteraction and binding on the molecular recognition layer by receivinginformation from an emitted fluorescent signal. An optical sensor of thesensor layer can be positioned to correspond with each independentlyaddressable location of the molecular recognition layer. In anembodiment, at least one or more optical sensors of the sensor layercorrespond to a probe or set of probes. In an embodiment, 1 opticalsensor corresponds to one independently addressable location. In anotherembodiment, 10-1000 optical sensors correspond to one independentlyaddressable location. In a further embodiment, different optical sensorscorresponding to the one independently addressable location measuredifferent wavelengths of light.

In an embodiment of the invention, the array of optical sensors of thesensor layer is a part of a semiconductor based sensor array. Thesemiconductor based sensor array can be either an organic semiconductoror an inorganic semiconductor. In some embodiments, the semiconductordevice is a silicon-based sensor. Examples of sensors useful in thepresent invention include, but are not limited to, a charge-coupleddevice (CCD), a CMOS device, and a digital signal processor. Thesemiconductor device of the sensor layer can also comprise an integratedin-pixel photocurrent detector. The detector may comprise a capacitivetransimpedance amplifier (CTIA).

In another embodiment, the semiconductor device has an in-pixel analogto digital converter.

In another embodiment, the array of optical sensors of the sensor layercan be a photodiode array.

The sensor layer can be created using a CMOS process. A semiconductordetection platform can be the assembly of an integrated system capableof measuring the binding events of real-time microarrays (RT-μArrays).In some embodiments, an integrated device system involves a transducerarray that is placed in contact with or proximity of the RT-μArrayassay.

A semiconductor detection platform for RT-μArrays can include an arrayof independent transducers to receive and/or analyze the signal fromtarget and probe binding events of a RT-μArray platform. A plurality oftransducers can work collectively to measure a number of binding eventsat any individual microarray spot. For example, transducers dedicated toa spot may add and/or average their individual measured signal.

Detection circuitry connected to an array of optical sensors can beembedded in the sensor layer. Signal processing circuitry can also beconnected to the array of optical sensors and embedded in the sensorlayer. In some embodiments, the transducers and/or detection circuitryand/or analysis systems are implemented using electronic componentswhich are fabricated and/or embedded in the semiconductor substrate.Examples of such fabrication techniques include, but are not limited to,silicon fabrication processes, micro-electromechanical surfacemicromachining, CMOS fabrication processes, CCD fabrication processes,silicon-based bipolar fabrication processes, and gallium-arsenidefabrication processes.

The transducer array can be an image sensor array. Examples of suchimage arrays include, but are not limited to, CMOS image sensor arrays,CMOS linear optical sensors, CCD image sensors, and CCD linear opticalsensors. The image sensor can be used to detect the activity of theprobe/analyte interaction within the integrated biosensor arrayplatform.

The pixel architecture within the CMOS image sensor array can be passivepixel sensor (PPS), active pixel sensor (APS) or digital pixel sensor(DPS) as illustrated in FIG. 4. In all these architectures the light inconverted to current using an integrated photodiode. The current in PPSis directly read from the photodiode by selecting the associated wordand bit lines, while in APS architecture the current is initiallyintegrated and converted to voltage and then is read. In DPS andanalog-to-digital converter digitizes the read signal making the pixeloutput digital as opposed to analog in PPS and APS.

As used herein, the terms detector and transducer are usedinterchangeably, and refer to a component that is capable of detecting asignal that can be correlated with an value of analyte/probe binding.

In some embodiments the detector array is in contact with the molecularrecognition layer. In some embodiments, the detector is spaced from themolecular recognition layer. The detector can be optically coupled tothe molecular recognition layer, for example, with one or more lenses orwaveguides.

FIG. 5 shows an example of a CMOS embodiment of the present invention. Asemiconductor is utilized as a sensor for detecting the binding of themolecular recognition layer, or in this example, the detection area. Thedetection area is located on the device in a layer separated from thesensor layer by an optical layer.

In some embodiments, the detector is optically coupled through imagingusing focal plane detector arrays: In this method the signal generatedfrom the system is focused on a focal point detector array. Thisapproach useful for optical detection systems where signal focusing canbe carried out using lenses and other optical apparatus. Examples ofdetectors in these embodiments are CMOS and CCD image sensors.

Detectors can be placed such that the signal generated from thecapturing region can only be observed by the dedicated detector. If amicroarray with multiple capturing spots is used, multiple detectors areused, each dedicated to an individual spot.

The detectors of the present invention are generally capable ofcapturing signal at multiple time points in real-time, during thebinding reaction. In some embodiments the detector is capable ofmeasuring at least two signals in less than about 1 psec, 5 psec, 0.1nsec, 0.05 nsec, 0.1 nsec, 0.5 nsec, 1 nsec, 5 nsec, 0.01 μsec, 0.05μsec, 0.1 μsec, 0.5 μsec, 1 μsec, 5 μsec, 0.01 msec, 0.05 msec, 0.1msec, 0.5 msec, 1 msec, 5 msec, 10 msec, 50 msec, 100 msec, 0.5 sec, 1sec, 5 sec, 10 sec, or 60 sec.

In some embodiments the detector detects the signal at the substrate. Insome embodiments the detector will detect the signal in the solution. Insome embodiments, the detector will detect signal in both the solutionand at the molecular recognition layer.

Where the detector is capable of detecting optical signals, the detectorcan be, for example a photomultiplier tube (PMT), a CMOS sensor, or aCCD sensor. In some embodiments, the detector comprises a fiber-opticsensor.

In some embodiments, the device comprising the sensor is capable ofsensitive fluorescent measurements including synchronous fluorimetry,polarized fluorescent measurements, laser induced fluorescence,fluorescence decay, and time resolved fluorescence.

Optical Layer

The biosensor arrays of the present invention will generally comprise anoptical layer. The fully integrated arrays of the present inventioncomprise an optical layer that at least comprises an optical filterlayer. The optical layer resides between the molecular recognition layerand the sensor layer, and transmits the light from the molecularrecognition layer to the sensor layer.

An optical layer of the biosensor array can comprises an optical filterlayer for transmitting a portion of the spectrum of light from themolecular recognition layer to the sensor layer. The optical filterlayer can be a band-pass, stop-band, and/or low-pass optical filter. Ina non-limiting example, the optical filter has a bandwidth of about 10nm to about 20 nm. In this context, the term bandwidth refers to therange of wavelengths that is either passed by a band pass filter, or therange of wavelengths that are stopped by a stop filter. In someembodiments, the optical filter is in the wavelength range of 400 nm to800 nm.

The choice of the wavelength characteristics of the optical filter layerdepends on the characteristics of the fluorescent moieties that areused. In some embodiments, a band-pass filter is used that cuts offlight corresponding to excitation, but allows transmission of lightcorresponding to emission of fluorescence. In some embodiments, theoptical filter attenuates fluorescent excitation light by a factor of10² to 10⁷. In some embodiments, the optical filter attenuatesfluorescent excitation light by a factor of 10³ to 10⁵.

In some embodiments, the optical filter is a multilayer dielectricfilter which covers the transducers.

In some embodiments of the invention with optical layers, the photonflux from the molecular recognition layer is guided to the transducerarray, using a plurality of optical waveguides that are of the opticalcoupling layer. The optical waveguides can operate to essentially mapthe emitted photon flux from the molecular recognition layer onto thetransducer array. The optical coupling layer can comprise a plurality ofoptical waveguides. Examples of signal coupling elements include fiberoptic cables, fiber optic bundles, fiber optic faceplates, and lightpipes. Thus, pluralities of transducers and/or spots are opticallyconnected to each other. In further embodiments of this invention, thewaveguide is a phaseplate system.

The optical layer can integrate the biochemical part of the microarraywith an emission filter and a photo-detector in CMOS where the molecularrecognition layer is immobilized on a planar surface of an opticallayer. The excitation photon flux tends to be orthogonal to the surfaceof the microarray, but the emission is in all directions. In order tocapture most of the emitted photons, an optical coupling layercomprising a fiber-optic faceplate (FOF) comprising densely packedoptical fibers can be positioned on top of the optical filter. The FOFprohibits light scattering and can guide a 2D fluorescence opticalpattern along the direction of its fibers. The FOF can be 2 μm to 20 cmthick. In some embodiments, the thickness of the FOF is in the range ofabout 5 μm to 1 cm. In an embodiment, the thickness of the FOF is 3 mm.The FOF thermally isolates the microarray assay from the CMOS die andphotodiodes as well as creating a distance between the aqueous solutionand the chip.

An example device of the invention comprising an FOF is illustrated inFIG. 6. The FOF is located below a capturing (or molecular recognitionlayer). When the fluorophores are bound to the capture layer, theemitted photon flux can travel through the fiber optics of the FOF inorder to send a greater amount of emitted photons to the sensor layerwithout allowing the excitation photon flux (F_(x)) through to thesensor layer. In this example, the FOF is 3 mm thick and the opticalfilter layer is 2.1 μm thick. The optical layer can transmit the photonflux from capture layer that has traveled through the FOF to a CMOSimage sensor layer. In this example, the CMOS sensor is about 1 mm thickand comprises structures (“metal curtains”) for directing the photonflux to a photodiode sensor embedded in the CMOS sensor layer. There aretwo photodiodes corresponding to the two independently addressablelocations on the molecular recognition layer of the example embodiment.Readout circuitry for transmitting the information captured by thephotodiodes is also embedded in the CMOS sensor layer.

As an example, the sensor array shown in FIG. 6 can be fabricated usinga standard digital 0.35 μm CMOS process in an area of 9 mm². The filteris fabricated using layer-by-layer deposition of dielectric materials tocreate a long-pass filter with transition wavelength of 560 nm on theFOF. The FOF is the cut to match the sensor array area and placed on topof the CMOS chip. To physically stabilize the FOF on the chip, anon-conductive epoxy is applied on the periphery. The surfacefunctionalization to immobilize the probe is then carried out on thesurface of the FOF. In some embodiments, the surface of the FOF can beSiO₂ and very similar to the surface of glass slides.

Systems

In an aspect of the invention, a biosensor system is disclosed thatcomprises an embodiment of a biosensor array of the invention, a lightsource that directs light to the biosensor array, a fluidic chamber forholding fluid in contact with the biosensor array, a temperaturecontroller for controlling the temperature of the fluid and/or themolecular recognition layer, and an interface that connects to thebiosensor array to allow electronic communication to and from the array.

The system typically comprises a light source, for example, forexcitation of fluorescence. The light source is generally opticallycoupled to the substrate, for example with one or more lenses orwaveguides. The light source can provide a single wavelength, e.g. alaser, or a band of wavelengths.

The light source that directs light to the biosensor array the lightsource can be fixed and illuminate some or all of the addressablelocations on a molecular recognition layer of the biosensor array.

In an embodiment, the system comprises a plurality of biosensor arrays,wherein at least two of the biosensor arrays are connected to the samecomputer. In a further embodiment, the system can comprise 2 to 12biosensor arrays, all of which are connected to the same computer. Thissystem allows for the measurement of binding on multiple arrayssimultaneously in one instrument using one computer system.

Control of temperature can be important to allow control of bindingreaction rates, e.g. by controlling stringency. The temperature can becontrolled by controlling the temperature at any place within the systemincluding controlling the temperature of the fluid or the temperature ofthe molecular recognition layer. Any temperature control can be used forcontrolling the temperature including, but not limited to, resistiveheaters, Peltier devices, infrared heaters, fluid flow, and gas flow.The temperatures can be the same or different for solution or substrateor different parts of each. In a preferable embodiment, the temperatureis consistent within the binding region. In some embodiments, thetemperature is controlled to within about 0.01, 0.05, 0.1, 0.5, or 1° C.

In some embodiments, the temperature can be rapidly changed during thebinding reaction. The system can be capable of changing the temperatureat a rate of temperature change corresponding to a change of 1° C. inless than about 0.01 msec, 0.1 msec, 0.5 msec, 1 msec, 5 msec, 10 msec,50 msec, 100 msec, 0.5 sec, 1 sec, 10 sec, or 60 sec.

In other embodiments, the temperature is changed slowly, graduallyramping the temperature over the course of the binding reaction.

An example of changing the temperature during the binding reactioninvolves a change in temperature to change the binding stringency andprobability. Many bindings in affinity-based biosensors are a strongfunction of temperature, thus by changing temperature, the stringencycan be altered, and the new capturing process can be observed with a newset of capturing probabilities.

The system can further be capable of measuring temperature at one ormultiple locations in the solution or on the biosensor array. Thetemperature can be measured by any means including, but not limited to,by thermometer, thermocouple, or thermochromic shift.

When temperature is measured, the system can utilize a feedback loop fortemperature control wherein the measured temperature is used as an inputto the system in order to more accurately control temperature.

A control system of a system of the invention can be a real-time controlsystem for reading the real-time measurements from a biosensor array ofthe invention.

FIG. 7 shows a block diagram of some of the components of systems of thepresent invention. The example system comprises (i) a biosensor arraywhich includes a molecular recognition layer comprising probes andanalytes, (ii) a light source for illuminating the molecular recognitionlayer of the biosensor array, (iii) heating and cooling modules and atemperature sensor, (iv) a temperature controller, and (v) a computer towhich sensor data is sent from the biosensor array. In some embodiments,the sensor data will be raw output from the optical sensors. In otherembodiments, the sensor data sent to the computer will be furtherprocessed by circuits within the optical sensor array.

The system can comprise a computing system for analyzing detectedsignals. In some embodiments, the system is capable of transferring timepoint data sets to the computing system wherein each time point data setcorresponds to detected signal at a time point, and the computing systemis capable of analyzing the time point data sets, in order to determinea property related to the analyte and probe. Thus, a computer system andsoftware that can store and manipulate the data (for instance, imagestaken at time points) may be components of the system. The data can beanalyzed in real-time, as the reaction unfolds, or may be stored forlater access.

The information corresponding to a detected signal at each time pointcan be single values such as signal amplitude, or can be more complexinformation, for example, where each set of signal informationcorresponds to an image of a region containing signal intensity valuesat multiple places within an addressable location.

The property related to analyte and/or probe can be, for example,analyte concentration, binding strength, or competitive binding, andcross-hybridization.

In some embodiments, the computing system uses algorithms fordetermining concentration and/or cross-hybridization.

The system can also comprise a computer with software for processcontrol and data collection and analysis. The software can be used forcharacterizing binding between analyte and probe. In one embodiment, thesoftware carries out at least one of the steps of i) accessing storedimages taken at different time points, ii) performing image processingto determine the location of the spots and convert the data to acollection of time series (one for each spot) representing the temporalbehavior of the signal intensity for each spot, and iii) for each spoton the array determining whether a reaction has happened (this is oftendone by comparing with control spots on the array). Optionally, thesoftware can perform the steps of iv) determining whether the reactionat each spot involves the binding of a single analyte or multipleanalytes (if, for example, cross-hybridization is occurring), v)estimating the reaction rates using statistical system identificationmethods. Examples of statistical system identification methods includemethods such as Prony's method. In the case that step iv) is used,(multiple bindings per spot), the reaction rate of each binding isdetermined, and vi) using the reaction rates to estimate the unknownquantity of interest (analyte concentration, binding strength, etc.)using, for example, optimal Bayesian methods.

In some embodiments, the system will have software for interfacing withthe instrument, for example, allowing the user to display information inreal-time and allowing for user to interact with the reaction (i.e., addreagents, change the temperature, change the pH, dilution, etc.).

The system can comprise control circuitry that enables users to activatereadout circuits and sensors on the array. The control circuitry canalso be used to specify temperature, mixing, fluorescent wavelength,fluorescent excitation power, pressure, and/or humidity. In addition,the control circuitry can be a part of the biosensor array.

In another embodiment, the biosensor system comprises decoder circuitryto select which of a plurality of sensors are to be used. At least partof the decoder circuitry can be part of the biosensor array. The decodercan select which of the plurality of transducers are to be used toanalyze the biosensor array probe. The control circuitry enables a userto activate a combination of read-out circuitries and transducers.Further, the system includes a function generator coupled to the controlcircuitry to generate the experiment control signals. The controlsignals are connected to systems inside and/or outside the semiconductorsubstrate. In certain embodiments, the control signal specifies themeasurement parameters. These parameters include, but are not limitedto, the biosensor array assay temperature, mixing speed, fluorescentexcitation signal wavelength, fluorescent excitation power, pressure,and humidity. In further embodiments the control signals are generatedby processing the measured signals. The processing can be carried outusing a digital signal processor outside the semiconductor substrateand/or an embedded digital signal processing unit inside thesemiconductor substrate.

In an embodiment of the invention illustrated in FIG. 8, a system cancomprise an assay kit, a disposable sensor chip, a computer chip readerand software for executing a method of the invention. The assay kit cancomprise the necessary reagents for sample preparation and labeling ofRT-μArray experiments. The sensor chip can comprise a CMOS chip withspotted capturing probes for measuring the level of hybridization inreal-time. The chip reader can be a dedicated reader for the disposablesensor chip and may or may not comprise a temperature controller,excitation signal generator, and data acquisition means.

Methods

The integrated biosensor array devices and systems of the invention canevaluate the abundance of a plurality of target analytes in the sampleby real-time detection of target-probe binding events. In certainembodiments, the integrated biosensor arrays are used as RT-μArraydetection systems to measure the concentration of the target analytes byanalyzing the binding rates and/or the equilibrium concentration and/orthe fluctuation of the captured analytes in a single and/or plurality ofspots. Applications of integrated biosensor array devices and systemsare within the field of Genomics and Proteomics, in particular DNA andProtein microarrays and immunoassays. In some embodiments, the discloseddevices, systems, and methods do not require any washing step and canalso measure the probe density variations prior to hybridization (thusallowing for pre-calibration of the experimental results). The RT-μArraysystems can also carry out various time averaging schemes to suppressthe Poisson noise and fluctuation of target bindings. Since targetconcentrations can be determined by the reaction rates, it is notnecessary for the hybridization process to go to equilibrium, resultingin faster detection times. A goal of the invention is to increase thedynamic range, sensitivity and resolution of microarrays. The inventionallows for quality control and may be more amenable to integration andto automation.

Advantages offered by the present invention can also apply to RNA,protein, carbohydrate, or lipid microarrays, to analyze interactionsamong molecules of the same or of different biochemical nature. Inparticular, real-time measurements by a platform of the invention, inwhich interactions are detected as they occur, allows for measuring andcomparing rates of analyte binding, affinities, etc. In addition, theRT-μArray can consist of microarrays in which compounds of two differentbiochemical classes (for example, protein and DNA, or carbohydrate andDNA) are positioned together as a mixture on the same spot of themicroarray.

The methods of the present invention can be used for measuring thebinding characteristics of multiple probes and analytes in real time.The method is particularly useful for characterizing the binding ofprobes and analytes which specifically bind to one another. As usedherein, a probe “specifically binds” to a specific analyte if it bindsto that analyte with greater affinity than it binds to other substancesin the sample.

The binding may be a receptor-ligand, enzyme-substrate,antibody-antigen, or a hybridization interaction. The probe/analytebinding pair or analyte/probe binding pair can be nucleic acid tonucleic acid, e.g. DNA/DNA, DNA/RNA, RNA/DNA, RNA/RNA, RNA. Theprobe/analyte binding pair or analyte/probe binding pair can be anucleic acid and a polypeptide, e.g. DNA/polypeptide andRNA/polypeptide, such as a sequence specific DNA binding protein. Theprobe/analyte binding pair or analyte/probe binding pair can be anynucleic acid and a small molecule, e.g. RNA/small molecule, DNA/smallmolecule. The probe/analyte binding pair or analyte/probe binding paircan be any nucleic acid and synthetic DNA/RNA binding ligands (such aspolyamides) capable of sequence-specific DNA or RNA recognition. Theprobe/analyte binding pair or analyte/probe binding pair can be aprotein and a small molecule or a small molecule and a protein, e.g. anenzyme or an antibody and a small molecule.

The probe/analyte binding pair or analyte/probe binding pair can be acarbohydrate and protein or a protein and a carbohydrate, a carbohydrateand a carbohydrate, a carbohydrate and a lipid, or lipid and acarbohydrate, a lipid and a protein, or a protein and a lipid, a lipidand a lipid.

The analyte/probe binding pair can comprise natural binding compoundssuch as natural enzymes and antibodies, and synthetic binding compounds.The probe/analyte binding pair or analyte/probe binding pair can besynthetic protein binding ligands and proteins or proteins and syntheticbinding ligands, synthetic carbohydrate binding ligands andcarbohydrates or carbohydrates and synthetic carbohydrate bindingligands, synthetic lipid binding ligands or lipids and lipids andsynthetic lipid binding ligands.

In an aspect of the invention, a method is described herein comprisingcontacting a fluid containing a target analyte with a biosensor array ofthe invention, illuminating the biosensor, detecting fluorescence on thebiosensor, and correlating the detected fluorescence with binding of thetarget analyte.

In an embodiment, a method of the invention comprises contacting a fluidvolume comprising a plurality of different analytes to a molecularrecognition layer of the biosensor array that comprises a plurality ofdifferent probes at independently addressable locations, wherein theprobes are capable of specifically binding to the analytes. The methodfurther comprises measuring signals at multiple time points while thefluid volume is in contact with the molecular recognition layer of abiosensor array, wherein the signals can be correlated with the amountof binding of the analytes with the probes. The biosensor array cancomprise an array of optical transducers, wherein at least one opticaltransducer correlates to an independently addressable location. A changein the signals with time can correlate with the amount of the analytesbound to the probes.

A fluid volume can be introduced and held in a system of the inventionby any method that will maintain the fluid in contact with the solidsupport. In many cases the fluid is held in a chamber. In someembodiments the chamber is open on one face, in other embodiments thechamber will mostly enclose the fluid. In some embodiments, the chamberwill have one or more ports for introducing and/or removing material(usually fluids) from the chamber. In some embodiments one side of thechamber comprises the solid substrate on which the probes are attached.In some embodiments the chamber is integral to the solid substrate. Insome embodiments, the chamber is a sub-assembly to which the solidsubstrate with probes can be removably attached. In some embodiments,some or all of the fluid chamber is an integral part of the device thatcomprises the detector. The chamber can be designed such that the signalthat can be correlated with analyte-probe binding can be detected by adetector outside of the chamber. For instance, all or a portion of thechamber can be transparent to light to allow light in or out of thechamber to facilitate excitation and detection of fluorophores.

In some embodiments, the means to perform the assay comprise acompartment wherein the surface of the microarray comprises a floor ofthe compartment and means to deliver reagents and analytes into thecompartment. Any method can be used to seal the microarray to thecompartment including using adhesives and gaskets to seal the fluid. Anymethod can be used to deliver reagents and analytes including usingsyringes, pipettes, tubing, and capillaries.

In some embodiments, the system comprises an apparatus to add or removematerial from the fluid volume. In some embodiments, the system can addor remove a liquid from the fluid volume. In some embodiments, thesystem is capable of adding or removing material from the fluid volumein order to change the: concentration, pH, stringency, ionic strength,or to add or remove a competitive binding agent. In some embodiments,the system is capable of changing the volume of the fluid volume duringthe reaction.

One exemplary embodiment of adding material to the fluid volume duringthe binding reaction comprises the addition of incubation buffer. Theincubation buffer is the buffer in which the analytes are residing. Byadding the incubation buffer, the concentration of analytes in thesystem will decrease and therefore the binding probability and kineticof binding will both decrease. Furthermore, if the reaction has alreadyreached equilibrium, the addition of the buffer will cause the system tomove another equilibrium state in time.

Another exemplary embodiment of adding material to the fluid volumeduring the binding reaction is adding a competing binding species. Thecompeting species can be of the same nature of the analyte but ingeneral they are molecules which have affinity to capturing probes. InDNA microarrays for example, the competing species can be synthesizedDNA oligo-nucleotides with partially or completely complementarysequence to the capturing probes. In immunoassays, the competing speciesare antigens.

In some embodiments, the system comprises elements to apply an electricpotential to the fluid volume to electrically change the stringency ofthe medium. In some embodiments, the system will provide an electricalstimulus to the capturing region using an electrode structure which isplaced in proximity of the capturing region. If the analyte is anelectro-active species and/or ion, the electrical stimulus can apply anelectrostatic force of the analyte. In certain embodiments, thiselectrostatic force is adjusted to apply force on the bonds betweenanalyte and capturing probe. If the force is applied to detach themolecule, the affinity of the analyte-probe interaction is reduced andthus the stringency of the bond is evaluated. The electrical stimulus isgenerally a DC and/or time-varying electrical potentials. Theiramplitude can be between 1 mV to 10V, but typically between 10 mV to 100mV. The frequency of time-varying signal can be between 1 Hz to 1000MHz, in some embodiments, the frequency of the time-varying signal isbetween 100 Hz to 100 kHz. The use of electric potential to controlstringency is described in U.S. Pat. No. 6,048,690.

The method can also further comprise using the signals measured atmultiple time points to determine the concentration of an analyte in thefluid volume.

In another aspect of a method of the invention, nucleic acidamplification can be performed on two or more nucleotide sequences witha device of the invention to produce two or more amplicons in a fluid. Asimilar integrated device for the methods of the invention capable ofperforming quantitative polymerase chain reactions is described in aco-pending U.S. patent application Ser. No. 11/829,861. The device cancomprise a solid surface with a plurality of nucleic acid probes atindependently addressable locations, wherein the solid substratecomprises an array of optical transducers, and at least one opticaltransducer correlates to an independently addressable location. Thehybridization of the amplicons to the two or more nucleic acid probescan also be measured while the fluid is in contact with the device toobtain an amplicon hybridization measurement. In a further embodiment,the method further comprises using the amplicon hybridizationmeasurement to determine the concentration of the amplicons in thefluid. In another embodiment the method further comprises using theamplicon hybridization measurement to determine the original amount ofnucleotide sequences.

In an embodiment, the method involves the use of probes in which eachaddressable location emits a signal that is quenchable upon binding ofan analyte. For example, the quenchable moiety (e.g., a fluorescentmoiety) is attached to the probe on the array or in close physicalproximity thereto. The surface of such array will emit signal from eachaddressable location which can be detected. The analytes in the sampleare tagged with a quencher moiety that can quench the signal from thequenchable moiety. When the quencher does not emit a light signal, thereis no signal from the fluid to interfere with the signal from the array.This diminishes the noise at the array surface. During the course of abinding reaction between analytes and substrate-bound probes, the signalat each addressable location can be quenched. The signal at eachaddressable location can be measured in real-time. As the signal at anylocation changes as a result of binding and quenching, the change ismeasured. These measurements over time allow determination of thekinetics of the reaction which, in turn, allows determination of theconcentration of analytes in the sample.

Alternatively if the analytes are labeled with a light-emittingreporter, such as a fluorescent label, signal at the surface of arrayresulting from binding of the labeled analyte molecules can be detectedby properly focusing the detector at the array surface, therebyminimizing the noise from signal in solution.

In another embodiment, the probes are attached to the surface of anarray comprising sensors, such as a CMOS sensor array, which produceelectrical signals that change as a result of binding events on theprobes. This also affords real-time measurement of a plurality ofsignals on an array (Hassibi and Lee, IEEE Sensors journal, 6-6, pp.1380-1388, 2006, and Hassibi, A. “Integrated Microarrays” Ph.D. ThesisStanford University, 2005).

Accordingly, the methods of this invention allow real-time measurementsof a plurality of binding events on an array of probes on a solidsupport.

Where the probe and analyte are nucleic acids, the present inventionprovides methods of expression monitoring and generic differencescreening. The term expression monitoring is used to refer to thedetermination of levels of expression of particular, typicallypreselected, genes. The invention allows for many genes, e.g. 10, 100,1,000, 10,000, 100,000 or more genes to be analyzed at once. Nucleicacid samples are hybridized to the arrays and the resultinghybridization signal as a function of time provides an indication of thelevel of expression of each gene of interest. In some embodiments, thearray has a high degree of probe redundancy (multiple probes per gene)the expression monitoring methods provide accurate measurement and donot require comparison to a reference nucleic acid.

In another embodiment, this invention provides generic differencescreening methods that identify differences in the abundance(concentration) of particular nucleic acids in two or more nucleic acidsamples. The generic difference screening methods involve hybridizingtwo or more nucleic acid samples to the same oligonucleotide array, orto different oligonucleotide arrays having the same oligonucleotideprobe composition, and optionally the same oligonucleotide spatialdistribution. The resulting hybridizations are then compared allowingdetermination which nucleic acids differ in abundance (concentration)between the two or more samples.

Where the concentrations of the nucleic acids comprising the samplesreflects transcription levels genes in a sample from which the nucleicacids are derived, the generic difference screening methods permitidentification of differences in transcription (and by implication inexpression) of the nucleic acids comprising the two or more samples. Thedifferentially (e.g., over- or under-) expressed nucleic acids thusidentified can be used (e.g., as probes) to determine and/or isolatethose genes whose expression levels differs between the two or moresamples.

The expression monitoring and difference screening methods of thisinvention may be used in a wide variety of circumstances includingdetection of disease, identification of differential gene expressionbetween two samples (e.g., a pathological as compared to a healthysample), screening for compositions that upregulate or down-regulate theexpression of particular genes, and so forth.

Analyte and Probe

The terms “probe” and “analyte” as used herein refer to molecularspecies that bind to one another in solution. A single probe or a singleanalyte is generally one chemical species. That is, a single analyte orprobe may comprise many individual molecules. In some cases, a probe oranalyte may be a set of molecules that are substantially identical. Insome cases a probe or analyte can be a group of molecules all of whichhave a substantially identical binding region. A “probe” and/or“analyte” can be any pair of molecules that bind to one another,including for example a receptor/ligand pair, or a hybridizing pair ofnucleic acids. In probes of the present invention are bound to a solidsurface. The analyte is in solution, and can also be referred to as thetarget or the target analyte. Thus, while the probe and analyte caninterchangeably be the different members of any binding pair, in somecases it is more advantageous for one or the other to be the probe orthe analyte, for instance where the molecule is more easily coupled tothe surface, it can be advantageous for that molecule to be the probe,or where a molecule is more soluble in the solution of interest, it canbe advantageous for that molecule to be the analyte.

The probes or analytes can be any type of chemical species. The probesor analytes are generally biomolecules such as nucleic acids, proteins,carbohydrates, lipids, or small molecules. The probe and analyte whichbind to one another can each be the same or different types of species.The analyte or probe may be bound to another type of molecule and maycomprise different molecules. For example, an analyte could be a proteincarbohydrate complex, or a nucleic acid connected to protein. Aprobe-analyte pair can also be a receptor-ligand pair. Where thechemical species is large or made of multiple molecular components, theprobe or analyte may be the portion of the molecule that is capable ofbinding, or may be the molecule as a whole. Examples of analytes thatcan be investigated by this invention include, but are not restrictedto, agonists and antagonists for cell membrane receptors, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),hormone receptors, peptides, enzymes, enzyme substrates, substrateanalogs, transition state analogs, cofactors, drugs, proteins,antibodies, and hybridizing nucleic acids.

The term “probe” is used herein to refer to the member of the bindingspecies that is attached to a surface. For instance, the probe consistsof biological materials deposited so as to create spotted arrays; andmaterials synthesized, deposited, or positioned to form arrays accordingto other current or future technologies. Thus, microarrays formed inaccordance with any of these technologies may be referred to generallyand collectively hereafter for convenience as “probe arrays.” Moreover,the term “probe” is not limited to probes immobilized in array format.Rather, the functions and methods described herein may also be employedwith respect to other parallel assay devices. For example, thesefunctions and methods may be applied with respect to probe-setidentifiers that identify probes immobilized on or in beads, opticalfibers, or other substrates or media. The construction of various probearrays of the invention are described in more detail below.

In some embodiments, the probe and/or the analyte comprises apolynucleotide. The terms “polynucleotide,” “oligonucleotide,” “nucleicacid” and “nucleic acid molecule” as used herein include a polymericform of nucleotides of any length, either ribonucleotides (RNA) ordeoxyribonucleotides (DNA). This term refers only to the primarystructure of the molecule. Thus, the term includes triple-, double- andsingle-stranded DNA, as well as triple-, double- and single-strandedRNA. It also includes modifications, such as by methylation and/or bycapping, and unmodified forms of the polynucleotide. More particularly,the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” include polydeoxyribonucleotides (containing2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any othertype of polynucleotide which is an N- or C-glycoside of a purine orpyrimidine base, and other polymers containing nonnucleotide backbones.A nucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (U.S. Pat. No. 5,644,048), phosphorodithioate(Briu et al., J. Am. Chem. Soc. 11 1:2321 (1989),O-methylphosphoroamidite linkages and peptide nucleic acid backbones andlinkages (see Carlsson et al., Nature 380:207 (1996)). Other analognucleic acids include those with positive backbones (Denpcy et al.,Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S.Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863,including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506.Nucleic acids containing one or more carbocyclic sugars are alsoincluded within the definition of nucleic acids. These modifications ofthe ribose-phosphate backbone may be done to facilitate the addition oflabels, or to increase the stability and half-life of such molecules inphysiological environments.

In some embodiments of the invention, oligonucleotides are used. An“oligonucleotide” as used herein is a single-stranded nucleic acidranging in length from 2 to about 1000 nucleotides, more typically from2 to about 500 nucleotides in length. In some embodiments, it is about10 to about 100 nucleotides, and in some embodiments, about 20 to about50 nucleotides.

In some embodiments of the invention, for example, expression analysis,the invention is directed toward measuring the nucleic acidconcentration in a sample. In some cases the nucleic acid concentration,or differences in nucleic acid concentration between different samples,reflects transcription levels or differences in transcription levels ofa gene or genes. In these cases it can be desirable to provide a nucleicacid sample comprising mRNA transcript(s) of the gene or genes, ornucleic acids derived from the mRNA transcript(s). As used herein, anucleic acid derived from an mRNA transcript refers to a nucleic acidfor whose synthesis the mRNA transcript or a subsequence thereof hasultimately served as a template. Thus, a cDNA reverse transcribed froman mRNA, an RNA transcribed from that cDNA, a DNA amplified from thecDNA, an RNA transcribed from the amplified DNA, etc., are all derivedfrom the mRNA transcript and detection of such derived products isindicative of the presence and/or abundance of the original transcriptin a sample. Thus, suitable samples include, but are not limited to,mRNA transcripts of the gene or genes, cDNA reverse transcribed from themRNA, cRNA transcribed from the cDNA, DNA amplified from the genes, RNAtranscribed from amplified DNA, and the like.

In the simplest embodiment, such a nucleic acid sample is the total mRNAor a total cDNA isolated and/or otherwise derived from a biologicalsample. The term “biological sample”, as used herein, refers to a sampleobtained from an organism or from components (e.g., cells) of anorganism. The sample may be of any biological tissue or fluid.Frequently the sample will be a “clinical sample” which is a samplederived from a patient. Such samples include, but are not limited to,sputum, blood, blood cells (e.g., white cells), tissue or fine needlebiopsy samples, urine, peritoneal fluid, and pleural fluid, or cellstherefrom. Biological samples may also include sections of tissues suchas frozen sections taken for histological purposes.

The nucleic acid (either genomic DNA or mRNA) may be isolated from thesample according to any of a number of methods well known to those ofskill in the art. One of skill will appreciate that where alterations inthe copy number of a gene are to be detected genomic DNA is preferablyisolated. Conversely, where expression levels of a gene or genes are tobe detected, preferably RNA (mRNA) is isolated.

Frequently, it is desirable to amplify the nucleic acid sample prior tohybridization. One of skill in the art will appreciate that whateveramplification method is used, if a quantitative result is desired, caremust be taken to use a method that maintains or controls for therelative frequencies of the amplified nucleic acids.

In some embodiments, the probe and or the analyte may comprise apolypeptide. As used herein, the term “polypeptide” refers to a polymerof amino acids and does not refer to a specific length of the product;thus, peptides, oligopeptides, and proteins are included within thedefinition of polypeptide. This term also does not refer to or excludepost expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. The“peptide” refers to polypeptides of no more than about 50 amino acids,while term “protein” refers to longer polypeptides, typically withthree-dimensional structures. Non-natural polypeptides containing one ormore analogs of an amino acid (including, for example, unnatural aminoacids, etc.), can also be useful in the invention, as can polypeptideswith substituted linkages, as well as other modifications known in theart, both naturally occurring and non-naturally occurring. Polypeptidesand proteins can have specific binding properties. For instance, anenzyme can have a region that binds specifically with a substrate,and/or has regions that bind to other proteins, such as the binding ofenzyme subunits. Antibodies, which can have very specific bindingproperties are also polypeptides.

In some embodiments the probe and/or analyte can comprise a carbohydratesuch as a polysaccharide. The term polysaccharide, as used herein,refers to a carbohydrate which is a polyhydroxy aldehyde or ketone, orderivative thereof, having the empirical formula (CH₂O)_(n) wherein n isa whole integer, typically greater than 3. Monosaccharides, or simplesugars, consist of a single polyhydroxy aldehyde or ketone unit.Monosaccharides include, but are not limited to, ribose, 2-deoxy-ribose,glucose, mannose, xylose, galactose, fucose, fructose, etc.Disaccharides contain two monosaccharide units joined by a glycosidiclinkage. Disaccharides include, for example, sucrose, lactose, maltose,cellobiose, and the like. Oligosaccharides typically contain from 2 to10 monosaccharide units joined in glycosidic linkage. Polysaccharides(glycans) typically contain more than 10 such units and include, but arenot limited to, molecules such as heparin, heparan sulfate, chondroitinsulfate, dermatan sulfate and polysaccharide derivatives thereof. Theterm “sugar” generally refers to mono-, di- or oligosaccharides. Asaccharide may be substituted, for example, glucosamine, galactosamine,acetylglucose, acetylgalactose, N-acetylglucosamine,N-acetyl-galactosamine, galactosyl-N-acetylglucosamine,N-acetylneuraminic acid (sialic acid), etc. A saccharide may also resideas a component part of a larger molecule, for example, as the saccharidemoiety of a nucleoside, a nucleotide, a polynucleotide, a DNA, an RNA,etc.

In some embodiments, the analyte and/or probe is a small molecule.Generally the small molecule will be an organic molecule, for example,biotin or digoxigenin, but in some cases, the analyte can be inorganic,for example an inorganic ion such as lithium, sodium, ferric, ferrous,etc. The small molecule can also be an organometallic compound, havingboth inorganic and organic components.

Optical Detection of Signals

For the methods of the present invention, a signal is detected that canbe correlated with the binding of analytes to the plurality of probes.The type of signals appropriate for the invention is any signal that canbe amount of analyte bound to the plurality of probes.

Examples of optical signals useful in the present invention are signalsfrom fluorescence, luminescence, and absorption. As used herein, theterms “electromagnetic” or “electromagnetic wave” and “light” are usedinterchangeably. Electromagnetic waves of any frequency and wavelengththat can be correlated to the amount of analyte bound to probe on thesurface can be used in the present invention including gamma rays,x-rays, ultraviolet radiation, visible radiation, infrared radiation,and microwaves. While some embodiments are described with reference tovisible (optical) light, the descriptions are not meant to limit theembodiments to those particular electromagnetic frequencies.

For the methods of the present invention it is desired that the signalchanges upon the binding of the analyte to the probe in a manner thatcorrelates with the amount of analyte bound. In some cases, the changein signal will be a change in intensity of the signal. In someembodiments, the signal intensity will increase as more analyte is boundto probe. In some embodiments, the signal intensity will decrease asmore analyte is bound to probe. In some embodiments, the change insignal is not a change in intensity, but can be any other change in thesignal that can be correlated with analyte binding to probe. Forexample, the change in signal upon binding of the probe can be a changein the frequency of the signal. In some embodiments, the signalfrequency will increase as more analyte is bound to probe. In someembodiments, the signal frequency will decrease as more analyte is boundto the probe.

The signal that is measured is generally the signal in the region of thesolid surface. In some embodiments, signal from moieties attached to thesurface is used as the signal that can be correlated with the amount ofanalyte bound to the probe. In some embodiments signal from the solutionis used as the signal that can be correlated with the amount of analytebound to the probe.

In some embodiments of the methods of the present invention, labels areattached to the analytes and/or the probes. Any label can be used on theanalyte or probe which can be useful in the correlation of signal withthe amount of analyte bound to the probe. It would be understood bythose of skill in the art that the type of label with is used on theanalyte and/or probe will depend on the type of signal which is beingused, for example, as described above, a dense label for a mechanicalsignal, or a redox active label for a voltammetric measurement.

In some embodiments, the signal that can be correlated to the amount ofanalyte bound to probe is due to the buildup of label at the surface asmore analyte is bound to the probes on the surface. For example, wherethe analyte has a fluorescent label, as more analyte binds, theintensity of the fluorescent signal can increase in a manner that can becorrelated with the amount of analyte bound to probe on the surface. Insome embodiments, the signal that can be correlated to the amount ofanalyte bound to probe is due to the release of label from the surface.For example, where the probe has a fluorescent label and the label isreleased into solution upon the binding of the analyte to the probe, thefluorescent intensity at the surface will decrease as more analyte isbound and more fluorescent label is released.

In some embodiments, the signal that can be correlated to the amount ofanalyte bound to probe is due to a change in the signal from label onthe surface upon binding of the analyte to the probe. For example, wherea fluorescent label is on the surface, and the analyte is labeled with acompound capable of changing the fluorescent signal of the surfacefluorescent label upon binding of the analyte with the probe, the changein signal can be correlated with the amount of analyte bound to probe.In some embodiments, the analyte is labeled with a quencher, and thedecrease in intensity from the surface fluorescent label due toquenching is correlated to the increased amount of analyte bound toprobe. In some embodiments, the analyte is labeled with a fluorescentcompound which can undergo energy transfer with the fluorescent label onthe surface such that the increase in fluorescence from the analytefluorescent label and/or the decrease in fluorescence from the surfacefluorescent label can be correlated with the amount of analyte bound toprobe. In some embodiments the surface fluorescent label is bounddirectly, e.g. covalently to the probe. In some embodiments, the surfacefluorescent label is bound to the surface, is not bound to the probe,but is in sufficient proximity that the binding of the analyte to theprobe produces a change in signal from the surface fluorescent labelthat can be correlated with the amount of analyte bound to probe.

In some embodiments, the analyte is unlabeled, and the bindingcharacteristics and or concentration of the analyte is determined bycompetitive binding with another labeled species, which competes withthe analyte for biding to a probe. For example, where we have a solutionwith an analyte, A, whose concentration we want to determine, and wehave a competitive binding species, B, whose binding characteristicswith probe and whose concentration are known, then using the presentinvention, we can use, for example, an array of probes on a surface todetermine the concentration of A by determining the amount ofcompetitive binding of B to a probe. For example, the probe is attachedto a surface that is fluorescently labeled, and B is labeled with aquencher such that the level of quenching of the surface fluorescencecan be correlated with the amount of B bound to the probe. The rate ofbinding of B to the probe is measured in real time, and theconcentration of A is determined by knowing the characteristics of A asa competitive binder. In some embodiments, the amount of the competitivebinding species does not need to be known beforehand. For instance, thekinetics of binding of be can be measured in the fluid volume, then theconditions can be changed, (e.g. increasing the stringency) such that Bis released from the probe, then the analyte A is added, and the bindingof B under competition with A is measured. This example illustrates anadvantage of the being able to change the conditions of the mediumduring one experiment. In some cases, A and B can be the same species,where B is labeled, and the amount of B is known, and the amount of Acan be determined by the kinetics of the binding of B. In some cases, Aand B are not the same species, but compete for binding with a probe.This competitive binding real-time assay can be done with all types ofmolecular species described herein including nucleic acids, antibodies,enzymes, binding proteins, carbohydrates and lipids.

Some embodiments of the invention involve measuring light absorption,for example by dyes. Dyes can absorb light within a given wavelengthrange allowing for the measurement of concentration of molecules thatcarry that dye. In the present invention, dyes can be used as labels,either on the analyte or on the probe. The amount of dye can becorrelated with the amount of analyte bound to the surface in order todetermine binding kinetics. Dyes can be, for example, small organic ororganometallic compounds that can be, for example, covalently bound tothe analyte to label the analyte. Dyes which absorb in the ultraviolet,visible, infrared, and which absorb outside these ranges can be used inthe present invention. Methods such as attenuated total reflectance(ATR), for example for infrared, can be used to increase the sensitivityof the surface measurement.

Some embodiments of the invention involve measuring light generated byluminescence. Luminescence broadly includes chemiluminescence,bioluminescence, phosphorescence, and fluorescence. In some embodiments,chemiluminescence, wherein photons of light are created by a chemicalreaction such as oxidation, can be used. Chemiluminescent species usefulin the invention include, without limitation, luminol, cyalume, TMAE(tetrakis(dimethylamino)ethylene), oxalyl chloride, pyrogallol(1,2,3-trihydroxibenzene), lucigenin. In some embodiments,bioluminescence is used. Where the luminescence is bioluminescence,creation of the excited state derives from an enzyme catalyzed reaction.Bioluminescence derives from the capacity of living organisms to emitvisible light through a variety of chemiluminescent reaction systems.Bioluminescence generally include three major components: a luciferin, aluciferase and molecular oxygen. However other components may also berequired in some reactions, including cations (Ca⁺⁺ and Mg⁺⁺) andcofactors (ATP, NAD(P)H). Luciferases are enzymes that catalyze theoxidation of a substrate, luciferin, and produce an unstableintermediate. Light is emitted when the unstable intermediate decays toits ground state, generating oxyluciferin. Any of the differentunrelated types of luciferin can be used herein including those fromphyla which use a luciferin, known as coelenterazine, which contains aring formed by three amino acids (2 tyrosines, and a phenylalanine).Photoproteins from animals such as jellyfish can be used where the“photoprotein” of the luciferin/luciferase system emits light uponcalcium binding. Other bioluminescent systems as described in U.S.Patent Application Publication No. 2007/0065818, and includingbioluminescence resonance energy transfer (BRET) as described in U.S.Patent Application Publication No. 2007/0077609 can be used in thepresent invention.

Fluorescent Systems

A useful embodiment of the present invention involves the use offluorescence. As used herein, fluorescence refers to the process whereina molecule relaxes to its ground state from an electronically excitedstate by emission of a photon. As used herein, the term fluorescencealso encompasses phosphorescence. For fluorescence, a molecule ispromoted to an electronically excited state by generally by theabsorption of ultraviolet, visible, or near infrared radiation. Theexcited molecule then decays back to the ground state, or to alower-lying excited electronic state, by emission of light. An advantageof fluorescence for the methods of the invention is its highsensitivity. Fluorimetry may achieve limits of detection several ordersof magnitude lower than for absorption. Limits of detection of 10⁻¹⁰ Mor lower are possible for intensely fluorescent molecules; in favorablecases under stringently controlled conditions, the ultimate limit ofdetection (a single molecule) may be reached.

A wide variety of fluorescent molecules can be utilized in the presentinvention including small molecules, fluorescent proteins and quantumdots. Useful fluorescent molecules (fluorophores) include, but are notlimited to: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA);5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-HydroxyTryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; Aequorin (Photoprotein); AFPs—AutoFluorescent Protein—(QuantumBiotechnologies); Texas Red; Texas Red-X conjugate; Thiadicarbocyanine(DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S;Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (CalcofluorWhite); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor(PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; TruRed;Ultralite; Uranine B; Uvitex SFC; WW 781; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; YO-PRO-1; YO-PRO-3; YOYO-1; YOYO-3, SybrGreen, Thiazole orange (interchelating dyes), or combinations thereof.

Some embodiments of the present invention include the Alexa Fluor dyeseries (from Molecular Probes/Invitrogen) which cover a broad spectrumand match the principal output wavelengths of common excitation sourcessuch as Alexa Fluor 350, Alexa Fluor 405, 430, 488, 500, 514, 532, 546,555, 568, 594, 610, 633, 635, 647, 660, 680, 700, and 750. Someembodiments of the present invention include the Cy Dye fluorophoreseries (GE Healthcare), also covering a wide spectrum such as Cy3, Cy3B,Cy3.5, Cy5, Cy5.5, Cy7. Some embodiments of the present inventioninclude the Oyster dye fluorophores (Denovo Biolabels) such asOyster-500, -550, -556, 645, 650, 656. Some embodiments of the presentinvention include the DY-Labels series (Dyomics), for example, withmaxima of absorption that range from 418 nm (DY-415) to 844 nm (DY-831)such as DY-415, -495, -505, -547, -548, -549, -550, -554, -555, -556,-560, -590, -610, -615, -630, -631, -632, -633, -634, -635, -636, -647,-648, -649, -650, -651, -652, -675, -676, -677, -680, -681, -682, -700,-701, -730, -731, -732, -734, -750, -751, -752, -776, -780, -781, -782,-831, -480XL, -481XL, -485XL, -510XL, -520XL, -521XL. Some embodimentsof the present invention include the ATTO fluorescent labels (ATTO-TECGmbH) such as ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590,594, 610, 611X, 620, 633, 635, 637, 647, 647N, 655, 680, 700, 725, 740.Some embodiments of the present invention include CAL Fluor and Quasardyes (Biosearch Technologies) such as CAL Fluor Gold 540, CAL FluorOrange 560, Quasar 570, CAL Fluor Red 590, CAL Fluor Red 610, CAL FluorRed 635, Quasar 670. Some embodiments of the present invention includequantum dots such as the EviTags (Evident Technologies) or quantum dotsof the Qdot series (Invitrogen) such as the Qdot 525, Qdot565, Qdot585,Qdot605, Qdot655, Qdot705, Qdot 800. Some embodiments of the presentinvention include fluorescein, rhodamine, and/or phycoerythrin.

FRET and Quenching

In some embodiments of the invention, fluorescence resonance energytransfer is used to produce a signal that can be correlated with thebinding of the analyte to the probe. FRET arises from the properties ofcertain fluorophores. In FRET, energy is passed non-radiatively over adistance of about 1-10 nanometers between a donor molecule, which is afluorophore, and an acceptor molecule. The donor absorbs a photon andtransfers this energy non-radiatively to the acceptor (Forster, 1949, Z.Naturforsch. A4: 321-327; Clegg, 1992, Methods Enzymol. 211: 353-388).When two fluorophores whose excitation and emission spectra overlap arein close proximity, excitation of one fluorophore will cause it to emitlight at wavelengths that are absorbed by and that stimulate the secondfluorophore, causing it in turn to fluoresce. The excited-state energyof the first (donor) fluorophore is transferred by a resonance induceddipole-dipole interaction to the neighboring second (acceptor)fluorophore. As a result, the excited state lifetime of the donormolecule is decreased and its fluorescence is quenched, while thefluorescence intensity of the acceptor molecule is enhanced anddepolarized. When the excited-state energy of the donor is transferredto a non-fluorophore acceptor, the fluorescence of the donor is quenchedwithout subsequent emission of fluorescence by the acceptor. In thiscase, the acceptor functions as a quencher.

Pairs of molecules that can engage in fluorescence resonance energytransfer (FRET) are termed FRET pairs. In order for energy transfer tooccur, the donor and acceptor molecules must typically be in closeproximity (up to 7 to 10 nanometers. The efficiency of energy transfercan falls off rapidly with the distance between the donor and acceptormolecules.

Molecules that can be used in FRET include the fluorophores describedabove, and includes fluorescein, 5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonicacid (EDANS). Whether a fluorophore is a donor or an acceptor is definedby its excitation and emission spectra, and the fluorophore with whichit is paired. For example, FAM is most efficiently excited by light witha wavelength of 488 nm, and emits light with a spectrum of 500 to 650nm, and an emission maximum of 525 nm. FAM is a suitable donorfluorophore for use with JOE, TAMRA, and ROX (all of which have theirexcitation maximum at 514 nm).

In some embodiments of the methods of the present invention, theacceptor of the FRET pair is used to quench the fluorescence of thedonor. In some cases, the acceptor has little to no fluorescence. TheFRET acceptors that are useful for quenching are referred to asquenchers. Quenchers useful in the methods of the present inventioninclude, without limitation, Black Hole Quencher Dyes (BiosearchTechnologies such as BHQ-0, BHQ-1, BHQ-2, BHQ-3, BHQ-10; QSY Dyefluorescent quenchers (from Molecular Probes/Invitrogen) such as QSY7,QSY9, QSY21, QSY35, and other quenchers such as Dabcyl and Dabsyl; Cy5Qand Cy7Q and Dark Cyanine dyes (GE Healthcare), which can be used, forexample, in conjunction with donor fluors such as Cy3B, Cy3, or Cy5;DY-Quenchers (Dyomics), such as DYQ-660 and DYQ-661; and ATTOfluorescent quenchers (ATTO-TEC GmbH), such as ATTO 540Q, 580Q, 612Q.

In some embodiments of the methods of the invention, both the analytesand the probes have labels that are members of a FRET pair, and thelabels are attached such that when an analyte binds to a probe, FRETwill occur between the labels, resulting in a change in signal that canbe correlated with the binding of analyte to probe in real-time. Thechange in signal can be the decrease in the intensity of the donorand/or the increase in the intensity of the acceptor. The FRET pair canbe chosen such that emission wavelength of the donor fluorophore is farenough from the emission wavelength of the acceptor fluorophore, thatthe signals can be independently measured. This allows the measurementof both the decrease in signal from the donor and the increase in signalfrom the acceptor at the same time, which can result in improvements inthe quality of the measurement of binding. In some cases, the probe willhave a label that is the donor of the donor-acceptor pair. In somecases, the analyte will have a label that is the donor of the donoracceptor pair.

In some embodiments of the methods of the invention, the analyte willhave a fluorescent label that is a member of a FRET pair, and the othermember of the FRET pair will be attached to the surface, wherein themember of the FRET pair attached to the surface is not covalently linkedto the probe. In some cases, the analyte will have a label that is thedonor of the donor-acceptor pair. In some cases, the analyte will have alabel that is the acceptor of the donor acceptor pair. In someembodiments, the member of the FRET pair that is attached to the surfaceis attached to an oligonucleotide which is attached to the surface (asurface-bound label). The oligonucleotide that is labeled with the FRETpair can be a nucleotide sequence that does not have a sequenceanticipated to specifically bind to an analyte. The use of asurface-bound label allows for the labeling of multiple areas of anarray without having to label each specific binding probe. This cansimplify the production of the array and reduce costs. We have foundthat even though the surface-bound FRET pairs are not covalently boundto the probe, they can be sensitive to the binding of the analytelabeled with the other member of the FRET pair in a manner that allowsthe change in signal to be correlated with the amount of analyte boundto probe.

In some embodiments of the methods of the present invention, the analyteis labeled with a quencher, and the probe is labeled with a donorfluorophore. The analyte is labeled with the quencher such that whenanalyte binds with the probe, the fluorescence from the fluorescentlabel on the probe is quenched. Thus, the signal, measured in real-time,can be correlated with the amount of binding of the analyte and theprobe, allowing for the measurement of the kinetics of the binding. Insome embodiments of the methods of the present invention, the analyte islabeled with a quencher, and the probe is labeled with a donorfluorophore, that is not covalently attached to it. The quencher islabeled such that when analyte binds with the probe, the fluorescencefrom the fluorescent label on the probe is quenched. Thus, the signal,measured in real-time, can be correlated with the amount of binding ofthe analyte and the probe, allowing for the measurement of the kineticsof the binding.

In some embodiments of the methods of the present invention, the analyteis labeled with a quencher, and the surface is labeled with a donorfluorophore wherein the donor fluorophore is not covalently linked tothe probe (e.g. with a surface bound fluorescent label). The quencher islabeled such that when analyte binds with the probe, the fluorescencefrom the fluorescent label on the surface is quenched. Thus, the signal,measured in real-time, can be correlated with the amount of binding ofthe analyte and the probe, allowing for the measurement of the kineticsof the binding.

Where the probe is labeled with a fluorophore, one aspect of theinvention is the use of an image of the fluorescently labeled probe onthe surface obtained before binding has occurred in order to effectivelyestablish a baseline signal for the state where no binding of analyte toprobe has occurred. In conventional arrays, in which unlabeled probe istreated with labeled analyte, and the signal is measured afterhybridization and washing, it can be difficult to know exactly how muchprobe is actually on the array in the region of interest. Thus,differences in array manufacture can affect the quality of the data. Inthe present invention, where the probe is labeled with fluorophore, theimage of the labeled probe on the surface provides a measurement of theamount of probe actually on the surface, increasing the quality andreliability of the binding measurement.

Nucleic Acid Systems

One particularly useful aspect of the present invention involvesspecific hybridization between an analyte and a probe, where bothcomprise nucleic acids.

As used herein an “oligonucleotide probe” is an oligonucleotide capableof binding to a target nucleic acid of complementary sequence throughone or more types of chemical bonds, usually through complementary basepairing, usually through hydrogen bond formation. The oligonucleotideprobe may include natural (i.e. A, G, C, or T) or modified bases(7-deazaguanosine, inosine, etc.). In addition, the bases inoligonucleotide probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, oligonucleotide probes may be peptide nucleic acidsin which the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. The oligonucleotide probes can also compriselocked nucleic acids (LNA), LNA, often referred to as inaccessible RNA,is a modified RNA nucleotide. The ribose moiety of the LNA nucleotide ismodified with an extra bridge connecting 2′ and 4′ carbons. The bridge“locks” the ribose in 3′-endo structural conformation, which is oftenfound in A-form of DNA or RNA. LNA nucleotides can be mixed with DNA orRNA bases in the oligonucleotide. Such oligomers are commerciallyavailable. The locked ribose conformation can enhance base stacking andbackbone pre-organization, and can increase the thermal stability(melting temperature) of oligonucleotides.

The term “nucleic acid analyte” or “target nucleic acid” or “target”refers to a nucleic acid (often derived from a biological sample andhence referred to also as a sample nucleic acid), to which theoligonucleotide probe specifically hybridizes. It is recognized that thetarget nucleic acids can be derived from essentially any source ofnucleic acids (e.g., including, but not limited to chemical syntheses,amplification reactions, forensic samples, etc.). It is either thepresence or absence of one or more target nucleic acids that is to bedetected, or the amount of one or more target nucleic acids that is tobe quantified. The target nucleic acid(s) that are detectedpreferentially have nucleotide sequences that are complementary to thenucleic acid sequences of the corresponding probe(s) to which theyspecifically bind (hybridize). The term target nucleic acid may refer tothe specific subsequence of a larger nucleic acid to which the probespecifically hybridizes, or to the overall sequence (e.g., gene or mRNA)whose abundance (concentration) and/or expression level it is desired todetect. The difference in usage will be apparent from context.

In the present invention, the specific hybridization of anoligonucleotide probe to the target nucleic acid can be measured inreal-time. An oligonucleotide probe will generally hybridize, bind, orduplex, with a particular nucleotide sequence under stringent conditionseven when that sequence is present in a complex mixture. The term“stringent conditions” refers to conditions under which a probe willhybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences.

For nucleic acid systems, the oligonucleotide probes of the presentinvention are designed to be complementary to a nucleic acid targetsequence, such that hybridization of the target sequence and the probesof the present invention occurs. This complementarity need not beperfect; there may be any number of base pair mismatches which willinterfere with hybridization between the target sequence and the singlestranded nucleic acids of the present invention. However, if the numberof mutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, an oligonucleotide probe that isnot substantially complementary to a nucleic acid analyte will nothybridize to it under normal reaction conditions.

The methods of the present invention thus can be used, for example, todetermine the sequence identity of a nucleic acid analyte in solution bymeasuring the binding of the analyte with known probes. The sequenceidentity can be determined by comparing two optimally aligned sequencesor subsequences over a comparison window or span, wherein the portion ofthe polynucleotide sequence in the comparison window may optionallycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical subunit (e.g.nucleic acid base or amino acid residue) occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity.

The methods of the current invention when applied to nucleic acids, canbe used for a variety of applications including, but not limited to, (1)mRNA or gene expression profiling, involving the monitoring ofexpression levels for example, for thousands of genes simultaneously.These results are relevant to many areas of biology and medicine, suchas studying treatments, diseases, and developmental stages. For example,microarrays can be used to identify disease genes by comparing geneexpression in diseased and normal cells; (2) comparative genomichybridization (Array CGH), involving the assessment of large genomicrearrangements; (3) SNP detection arrays for identifying for SingleNucleotide Polymorphisms (SNP's) in the genome of populations; andchromatin immunoprecipitation (chIP) studies, which involve determiningprotein binding site occupancy throughout the genome, employingChIP-on-chip technology.

The present invention can be very sensitive to differences in bindingbetween nucleic acid species, in some cases, allowing for thediscrimination down to a single base pair mismatch. And because thepresent invention allows the simultaneous measurement of multiplebinding events, it is possible to analyze several speciessimultaneously, where each is intentionally mismatched to differentdegrees. In order to do this, a “mismatch control” or “mismatch probe”which are probes whose sequence is deliberately selected not to beperfectly complementary to a particular target sequence can be used, forexample in expression arrays. For each mismatch (MM) control in an arraythere, for example, exists a corresponding perfect match (PM) probe thatis perfectly complementary to the same particular target sequence. In“generic” (e.g., random, arbitrary, haphazard, etc.) arrays, since thetarget nucleic acid(s) are unknown, perfect match and mismatch probescannot be a priori determined, designed, or selected. In this instance,the probes can be provided as pairs where each pair of probes differs inone or more pre-selected nucleotides. Thus, while it is not known apriori which of the probes in the pair is the perfect match, it is knownthat when one probe specifically hybridizes to a particular targetsequence, the other probe of the pair will act as a mismatch control forthat target sequence. It will be appreciated that the perfect match andmismatch probes need not be provided as pairs, but may be provided aslarger collections (e.g., 3, 4, 5, or more) of probes that differ fromeach other in particular preselected nucleotides. While the mismatch(s)may be located anywhere in the mismatch probe, terminal mismatches areless desirable as a terminal mismatch is less likely to preventhybridization of the target sequence. In a particularly preferredembodiment, the mismatch is located at or near the center of the probesuch that the mismatch is most likely to destabilize the duplex with thetarget sequence under the test hybridization conditions. In aparticularly preferred embodiment, perfect matches differ from mismatchcontrols in a single centrally-located nucleotide.

It will be understood by one of skill in the art that control of thecharacteristics of the solution such as the stringency are important inusing the present invention to measure the binding characteristics of aanalyte-probe pair, or the concentration of a nucleic acid analyte(target nucleic acid). A variety of hybridization conditions may be usedin the present invention, including high, moderate and low stringencyconditions; see for example Maniatis et al., Molecular Cloning: ALaboratory Manual, 2d Edition, 1989, and Short Protocols in MolecularBiology, ed. Ausubel, et al, hereby incorporated by reference. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, “Overview of principlesof hybridization and the strategy of nucleic acid assays” (1993). Insome embodiments, highly stringent conditions are used. In otherembodiments, less stringent hybridization condition; for example,moderate or low stringency conditions may be used, as known in the art;see Maniatis and Ausubel, supra, and Tijssen, supra. The hybridizationconditions may also vary when a non-ionic backbone, i.e. PNA is used, asis known in the art.

Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences tend to hybridize specificallyat higher temperatures. Generally, stringent conditions can be selectedto be about 5.degree. C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength, pH, and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. (As the targetanalyte sequences are generally present in excess, at T_(m), 50% of theprobes are occupied at equilibrium). Typically, stringent conditionswill be those in which the salt concentration is at least about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

In some embodiments, the probe and or the analyte may comprise anantibody. As used herein, the term “antibody” refers to animmunoglobulin molecule or a fragment of an immunoglobulin moleculehaving the ability to specifically bind to a particular molecule,referred to as an antigen. The antibody may be an anti-receptor antibodyspecific for the receptor used in the assay. Thus, the antibody may becapable of specifically binding the receptor as the antigen. Antibodiesand methods for their manufacture are well known in the art ofimmunology. The antibody may be produced, for example, by hybridoma celllines, by immunization to elicit a polyclonal antibody response, or byrecombinant host cells that have been transformed with a recombinant DNAexpression vector that encodes the antibody. Antibodies include but arenot limited to immunoglobulin molecules of any isotype (IgA, IgG, IgE,IgD, IgM), and active fragments including Fab, Fab′, F(ab′)₂, Facb, Fv,ScFv, Fd, V_(H) and V_(L). Antibodies include but are not limited tosingle chain antibodies, chimeric antibodies, mutants, fusion proteins,humanized antibodies and any other modified configuration of animmunoglobulin molecule that comprises an antigen recognition site ofthe required specificity.

The preparation of antibodies including antibody fragments and othermodified forms is described, for example, in “Immunochemistry inPractice,” Johnstone and Thorpe, Eds., Blackwell Science, Cambridge,Mass., 1996; “Antibody Engineering,” 2nd edition, C. Borrebaeck, Ed.,Oxford University Press, New York, 1995; “Immunoassay”, E. P. Diamandisand T. K. Christopoulos, Eds., Academic Press, Inc., San Diego, 1996;“Handbook of Experimental Immunology,” Herzenberg et al., Eds, BlackwellScience, Cambridge, Mass., 1996; and “Current Protocols in MolecularBiology” F. M. Ausubel et al., Eds., Greene Pub. Associates and WileyInterscience, 1987, the disclosures of which are incorporated herein. Awide variety of antibodies also are available commercially.

In some embodiments, the probe and or the analyte may comprise twoproteins. Protein-protein interactions can enable two or more proteinsto associate. A large number of non-covalent bonds can form between theproteins when two protein surfaces are precisely matched, and thesebonds account for the specificity of recognition. Protein-proteininteractions are involved, for example, in the assembly of enzymesubunits; of multiprotein enzymatic complexes, or of molecular machines;in enzyme-substrate reactions; in antigen-antibody reactions; in formingthe supramolecular structures of ribosomes, filaments, and viruses; intransport; and in the interaction of receptors on a cell with growthfactors and hormones. Products of oncogenes can give rise to neoplastictransformation through protein-protein interactions. For example, someoncogenes encode protein kinases whose enzymatic activity on cellulartarget proteins leads to the cancerous state. Another example of aprotein-protein interaction occurs when a virus infects a cell byrecognizing a polypeptide receptor on the surface, and this interactionhas been used to design antiviral agents. In some cases, protein-proteininteractions can be dependent on protein modifications. For example,histone proteins can be modified at different positions with differentchemical tags (e.g. phosphorylation, or methylation), and themodifications themselves be required or involved in the recognition byother proteins (e.g chromatin remodeling and associated proteins).

Example 1

This example illustrates how an optical filter layer in the form of aband-pass filter on the surface of the CMOS chip enhances opticalperformance of the integrated biosensor array. A challenge of designingany fluorescent-based detector is the proper optical excitation anddetection of fluorescent labels. FIG. 9 illustrates the absorption andemission spectra of Cy3 molecule which is an example fluorophore of asystem. The absorbed photon density for Cy3, denoted by A, exposed tothe incident photon flux, F_(X), obeys the Beer-Lambert law. For a thinlayer of diluted absorbing media with Cy3 as in microarray applicationswe haveA=F _(X)[1−e ^(−a) ⁰ ^((λ)N) ]≈F _(X) a ₀(λ)N,  (1)where a₀(λ) and N are the extinction coefficient in wavelength λ andsurface concentration of Cy3 respectively. Considering Q_(Y), thefluorescence quantum yield of Cy3, we can calculate I_(E), the totalemitted photons per surface area byI _(E) =Q _(Y) A≈Q _(Y) F _(X) a ₀(λ)N  (2)

As evident in FIG. 9, the photon emission is, to first order,proportional to N which is parameter of interest in microarrays. Themajor impediment for measuring I_(E) and therefore N, is the presence ofF_(X) during detection. Although F_(X) has a different wavelength fromI_(E), is very typical for F_(X) to be to 4-5 orders of magnitude largerthan I_(E) in microarray applications. To block F_(X) in our system, amulti-layer dielectric Fabry-Perot optical band-pass filter has beenfabricated on the surface of a CMOS chip. This emission filter can blockF_(X) by 100 dB in the Cy3 excitation band of 545-550 nm, while onlyloosing 25% of the signal in the pass-band.

Example 2

This example describes the construction of a fully integrated biosensorarray of the present invention. To design the CMOS photo-detector aNwell/Psub photodiode array is used in the 0.35 μm CMOS process. Eachdiode is 50 μm×50 μm and the array pitch is 250 μm. This dimension iscompatible with commercial microarray specifications and also minimizesthe optical cross-talk between photodiodes while providing sufficientspace to integrate in-pixel a photocurrent detector and an analog todigital converter (ADC).

Most of the CMOS image sensors use direction integration, where thephotocurrent is directly integrated over a reverse bias photodiodecapacitor. In the design, a capacitive transimpedance amplifier (CTIA)in the pixel is used as a photocurrent integrator. Comparing with aCTIA, a direct integrator suffers from the junction capacitancevariation as the reverse bias voltage changes depending on the outputsignal level. A CTIA does not have any such problems since thephotodiode bias is regulated by an operational transimpedance amplifier(OTA) used in the CTIA and it is set to VR1 as shown in FIG. 10.Photocurrent input in the system is integrated using the feedbackcapacitor.

In order to take full advantage of integration capability of CMOS, anADC is also included in the pixel level. Pixel level processing relaxesthe speed requirement of ADC while removing all analog signal bus linesin the array and significantly reduces the cross-talk issues. Tosuppress the low frequency noise of the comparator, a chopper stabilizedpreamplifier has been implemented with overall voltage gain ofapproximately 60 dBgain.

A measurement of the concentration of Cy3 fluorophores on the surface ofthe chip using our integrated microarray system can be performed todetermine the performance compatibility of this system withfluorescent-based microarrays.

FIG. 11 shows the die photo and the I/O pin allocation of the integrated7 by 8 pixel biosensor array. Auxiliary circuits beside the biosensorarray include row and column decoder and column switches which arenecessary for the functionality of the array.

What is claimed is:
 1. An integrated biosensor array, comprising, inorder, a molecular recognition layer, an optical layer, and a sensorlayer integrated in a sandwich configuration, wherein: (a) the molecularrecognition layer comprises a plurality of different probes attached atdifferent independently addressable locations, each of saidindependently addressable locations configured to receive an excitationphoton flux directly from a single source located on a single side ofsaid molecular recognition layer, wherein the molecular recognitionlayer transmits light to the optical layer; (b) the optical layercomprises an optical filter layer, wherein the optical layer transmitslight from the molecular recognition layer to the sensor layer, wherebythe transmitted light is filtered; and (c) the sensor layer comprises anarray of optical sensors that detect the filtered light transmittedthrough the optical layer, said sensor layer comprising sensor elementsfabricated using a CMOS fabrication process; wherein said molecularrecognition layer, said optical layer and said sensor layer comprise anintegrated structure in which said molecular layer is in contact withsaid optical layer and said optical layer is in contact with said sensorlayer.
 2. The biosensor array of claim 1 wherein the optical layerfurther comprises an optical coupling layer between the optical filterlayer and the molecular recognition layer.
 3. The biosensor array ofclaim 2 wherein the optical coupling layer comprises a plurality ofoptical waveguides.
 4. The biosensor array of claim 2 wherein theoptical coupling layer comprises a fiber-optic faceplate.
 5. Thebiosensor array of claim 1 wherein the optical layer is 2 μm to 20 cmthick.
 6. The biosensor array of claim 1 wherein the optical layer is 5μm to 1 cm thick.
 7. The biosensor array of claim 1 wherein the opticallayer provides thermal insulation between the array of optical sensorsand the molecular recognition layer.
 8. The biosensor array of claim 1wherein the sensor layer comprises a photodiode array.
 9. The biosensorarray of claim 1 wherein the sensor layer comprises embedded detectioncircuitry connected to the optical sensors.
 10. The biosensor array ofclaim 1 wherein the sensor layer comprises embedded detection and signalprocessing circuitry connected to the optical sensors.
 11. The biosensorarray of claim 1, wherein the integrated biosensor array comprises adigital signal processor.
 12. The biosensor array of claim 1, whereinthe integrated biosensor array comprises an integrated in-pixelphotocurrent detector.
 13. The biosensor array of 12 wherein theintegrated in-pixel photocurrent detector comprises a capacitivetransimpedance amplifier (CTIA).
 14. The biosensor array of claim 1,wherein the integrated biosensor array has an in-pixel analog to digitalconverter.
 15. The biosensor array of claim 1 wherein the optical filterlayer comprises a multilayer dielectric.
 16. The biosensor array ofclaim 15 wherein the optical filter layer has a passband or a stopbandwith a bandwidth of about 10 nm to 20 nm.
 17. The biosensor array ofclaim 16 wherein the passband or stopband is in the range of 400 nm to800 nm.
 18. The biosensor array of claim 1 wherein the optical filterlayer attenuates fluorescent excitation light by 10² to 10⁷.
 19. Thebiosensor array of claim 1 wherein the optical filter layer attenuatesfluorescent excitation light by 10³ to 10⁵.
 20. The biosensor array ofclaim 1 wherein at least one optical sensor corresponds to oneindependently addressable location comprising a probe.
 21. The biosensorarray of claim 1 wherein more than one optical sensor corresponds to oneindependently addressable location.
 22. The biosensor array of claim 1wherein about 10 to about 1000 optical sensors correspond to oneindependently addressable location.
 23. The biosensor array of claim 1wherein different optical sensors corresponding to one independentlyaddressable location measure different wavelengths of light.
 24. Thebiosensor array of claim 1 wherein the molecular recognition layercomprises 2 to 1,000,000 probes.
 25. The biosensor array of claim 1wherein the independently addressable locations comprise probescomprising fluorescent moieties.
 26. The biosensor array of claim 25wherein the fluorescent moieties are capable of being quenched uponbinding of an analyte comprising a quencher.
 27. The biosensor array ofclaim 25 wherein the fluorescent moieties are bound to the probes. 28.The biosensor array of claim 25 wherein the fluorescent moieties arebound to the surface of the array, but are not covalently bound to theprobes.
 29. The biosensor array of claim 1 wherein the probes comprisenucleic acids.
 30. The biosensor array of claim 1 wherein the probescomprise proteins.